EA003900B1 - Truncated tumor necrosis factor receptors, pharmaceutical composition on their base and methods os use of said factors and compositions - Google Patents

Abstract

1. A truncated sTNFR having the following formula: R1- [Cys<19>-Cys<103>] -R2 wherein [Cys<19>-Cys<103>] represents residues 19 through 103 of sTNFR-I, the amino acid residue numbering scheme of which is provided in Figure 1 (SEQ ID NO:2) to facilitate the comparison; wherein R1 represents a methionylated or nonmethionylated amine group of Cys<19> or of aminoterminus amino acid residue(s) selected from the group: and wherein R2 represents a carboxy group of Cys<103> or of carboxy-terminal amino acid residues, selected from the group: and variants and derivatives thereof, provided however, when R1 represents a methionylated or nonmethionylated amine group of amino acid sequence VCPQGKYIHPQNNSIC or an N-terminal truncation thereof of from 1 to 15 residues, then R1-[Cys<19>-Cys<103>]-R2 is not an addition variant having the formula R1-[Cys<19>-Cys<103>]-FCCSLCL-R3, wherein R3 represents a carboxyl group of amino acid residues Asn<111>-Asn<161> of Figure 1 or a carboxy-terminal truncation of Asn<111>-Asn<161> of Figure 1. 2. The tumor necrosis binding protein according to Claim 1, selected from the group consisting of 3. A truncated sTNFR having the following formula: R4-[Cys<32>-Cys<115>]-R5 wherein [Cys<32>-Cys<115>] represents amino acid residues Cys<32> through Cys<115> of sequence SEQ ID NO:35 provided in Figure 8; wherein R4 represents a methionylated or nonmethionylated amine group of Cys<32> or of aminoterminus amino acid residue(s) selected from the group, comprising and wherein R5 represents a carboxy group of Cys<115> or of carboxy-terminal amino acid residues selected from the group, comprising and variants thereof, provided however, when R4 represents a methionylated or non-methionylated amine group of amino acid sequence TCRLREYYDQTAQMC or an N-terminal truncation thereof of from 1 to 15 residues, then R4-[Cys<32>-Cys<115>]-R5 is not an addition variant having the formula R4-[Cys<32>-Cys<115>]-APLRKCR-R6, wherein R6 represents a carboxyl group of amino acid residues Pro<123>-Thr<179> of Figure 8 or a carboxy-terminal truncation of Pro<123>-Thr<179> of Figure 8. 4. The truncated sTNFR according to any one of Claims 1 - 3, wherein it has non-glycosylated amino-acid sequence. 5. The truncated sTNFR according to any one of Claims 1 - 3, wherein it has glycosylated amino acid sequence. 6 The truncated sTNFR according to any one of Claims 1 - 3, wherein it is conjugated to a water-soluble polymer. 7. The truncated sTNFR according to Claim 6, wherein the water-soluble polymer is polyethylene glycol. 8. A polyvalent sTNFR comprising at least one of receptors according to any one of Claims 1 though 7. 9. A polyvalent sTNFR according to claim 8, wherein it contains a dimmer of truncated receptor of claim 3. 10. A polyvalent tumor necrosis binding protein having the formula R1-X-R2, wherein: X comprises a linker, wherein said linker is a water soluble polymer; and R1 and R2 are biologically-active molecules covalently bonded to said water soluble polymer, wherein at least one of R1 and R2 is a tumor necrosis binding protein according to any one of Claims 1 - 5. 11. The polyvalent receptor of Claim 10, wherein the water-soluble polymer is polyethylene glycol. 12. The polyvalent receptor of Claim 11, wherein the truncated receptor is selected from the group consisting of sTNFR-I 2.6D/C105db and sTNFR-I 2.6D/C106db. 13. A polynucleotide encoding the sTNFR according to any one of Claims 1-3, selected from group comprising: polynucleotide with sequence as shown in Fig. 2 (SEQ ID NO: 3); polynucleotide with sequence as shown in Fig. 3 (SEQ ID NO: 5); polynucleotide with sequence as shown in Fig. 4 (SEQ ID NO: 7); polynucleotide with sequence as shown in Fig. 5 (SEQ ID NO: 11); polynucleotide with sequence as shown in Fig. 6 (SEQ ID NO: 9); polynucleotide with sequence as shown in Fig. 7 (SEQ ID NO: 13); polynucleotide with sequence which is degenerated in the coding regions or portions thereof of said sequences. 14. A vector comprising a polynucleotide of any one of Claims 13 through 15 operatively linked to an expression control sequence. 15. A prokaryotic host cell containing a polynucleotide of Claim 13 or a vector of Claim 14. 16. A eukaryotic host cell containing a polynucleotide of Claim 13 or a vector of Claim 14. 17. A method of preparation of truncated sTNFR providing the growing host cells of Claims 15 or 16 in conditions, providing the expression of said truncated sTNFR by host cells, and isolation of it. 18. The method according to Claim 17, wherein said host cells are E. coli. 19. The method according to Claim 17, wherein said host cells are Chinese hamster ovary cells. 20. Truncated sTNFR according to any of claims 1-7, characterized in that it is prepared by recombinant method. 21. A method of preparing of pharmaceutical composition wherein a therapeutically effective amount of the tumor necrosis factor receptor according to any one of Claims 1 -7 or Claim 20 or a polyvalent tumor necrosis factor receptor according to Claims 8-12 is mixed with one or more pharmaceutically acceptable vehicles. 22. A pharmaceutical composition comprising the tumor necrosis factor receptor according to any of Claims 1-7 or 20 in association with a pharmaceutically acceptable vehicle. 23. A pharmaceutical composition comprising the polyvalent tumor necrosis factor receptor according to any of Claims 8-12 in association with a pharmaceutically acceptable vehicle. 24. A pharmaceutical composition according to Claims 22 or 23, wherein it is continuous release composition. 25. A pharmaceutical composition according to Claims 22 or 23, wherein it is lyophilized. 26. A method of treating a TNF-mediated disease comprising administering to a patient the truncated sTNFR according to any of Claims 1-7 or 20 or polyvalent receptor according to any of Claims 8-12. 27. The method of claim 26, wherein the TNF-mediated disease is diabetes, hyperalgesia, inflammatory disease of bowels, ischemic and reperfusion injuries and rheumatic disease. 28. The method of claim 27, wherein a rheumatic disease is selected from group comprising arthritis, osteoarthritis, juvenile arthritis, ankylosing spondylitis, dermatomyositis, psoriatic arthritis, sclerodermatitis, Sjogren's sicca syndrome and vasculitis. 29. The method according to any of Claims 26-28, wherein the truncated or polyvalent sTNFR are administered intravenously, intramuscularly, intracutaneously, subcultaneously, intraarticular, or by injection. 30. The method according to any of Claims 26-29, wherein the truncated or polyvalent sTNFR are administered before, in combination and after administering of anti-inflammatory treatment. 31. The method according to Claim 30, wherein an anti-inflammatory treatment is selected from the group, comprising non-steroid anti-inflammatory treatments corticosteroids, slow antirheumatic preparations, and preparations modifying the disease. 32. The method according to Claim 30, wherein an anti-inflammatory treatment is interleikine-1 inhibitor (IL-1), selected from antagonist receptor of interleikine-1 (IL-ra) or soluble IL-1 receptor. 33. Use of truncated sTNFR according to Claims 1-7 or 20 or polyvalent receptor of Claims 8-12 for preparing of preparation for treating TNF-mediated disease. 34. Use of truncated sTNFR according to Claims 1-7 or 20 or polyvalent receptor of Claims 8-12 for preparing of preparation for treating diabetes, hyperalgesia, inflammatory disease of bowels, ischemic and reperfusion injuries and rheumatic disease. 35. Use of Claim 34, characterized in that rheumatic disease is selected from group comprising arthritis, osteoarthritis, juvenile arthritis, ankylosing spondylitis, dermatomyositis, psoriatic arthritis, sclerodermatitis, Sjogren's sicca syndrome and vasculitis. 36. The method according to any of Claims 33-35, wherein the said preparation is administered intravenously, intramuscularly, intracutaneously, subcultaneously, intraarticular, or by injection. 37. The method according to any of Claims 33-36, wherein the said preparation sTNFR is administered before, in combination and after administering of anti-inflammatory treatment. 38. The use according to Claim 37, wherein an anti-inflammatory treatment is selected from the group, comprising non-steroid anti-inflammatory treatments corticosteroids, slow antirheumatic preparations, and preparations modifying the disease. 39. The use according to Claim 37, wherein an anti-inflammatory treatment is interleikine-1 inhibitor (IL-1), selected from antagonist receptor of interleikine-1 (IL-ra) or soluble IL-1 receptor. 40. A kit for preparing an aqueous protein formulation comprising a first container with tumor necrosis factor receptor according to any one of Claims 1-7 and a second container having a physiologically acceptable solvent. 41. The method according to Claim 31, wherein an anti-inflammatory treatment is methotrexate. 42. Use according to Claim 38, wherein an anti-inflammatory treatment is methotrexate. 43. Pharmaceutical composition, according to any of Claims 22-25, wherein it further comprises an anti-inflammatory treatment. 44. Pharmaceutical composition according to Claim 44, wherein an anti-inflammatory treatment is selected from the group, comprising non-steroid anti-inflammatory treatments corticosteroids, slow antirheumatic preparations, and preparations modifying the disease. 45. Pharmaceutical composition according to Claim 44, wherein an anti-inflammatory treatment is methotrexate. 46. Pharmaceutical composition according to Claim 43, wherein an anti-inflammatory treatment is interleikine-1 inhibitor (IL-1), selected from antagonist receptor of interleikine-1 (IL-ra) or soluble IL-1 receptor. 47. Pharmaceutical composition according to Claim 46, wherein the antagonist receptor of interleikine-1 (IL-ra) comprises a sequence of human protein IL-1ra.

Description

The present invention relates to the field of treatment of inflammation. More specifically, the present invention relates to truncated receptors for tumor necrosis factor (δΤΝΡΚδ).

BACKGROUND OF THE INVENTION

Inflammation is a defensive reaction of the body in response to mechanical damage, infection, or antigenic stimulation. The inflammatory reaction can proceed according to the pathological type; when it is triggered by an inadequate stimulus, such as an autoantigen, it develops in an unusual way or continues after the elimination of the damaging agents.

Despite the fact that the etiology of inflammation is poorly studied, a large amount of information on the molecular aspects of inflammation has recently been obtained. These studies have led to the identification of individual cytokines that are actively involved in mediating inflammation. Cytokines are called extracellular proteins that modify the behavior of cells, especially those cells that are located near the area of synthesis and release of cytokines. Tumor necrosis factors (ΤΝΡδ) are a class of cytokines produced by various types of cells, including monocytes and macrophages.

At least two ΤΝΡδ, in particular, аль-alpha (ΤΝΡ-α) and ΤΝΡ beta (ΤΝΡ-β or lymphotoxin), each of which is active in the form of a three-dimensional molecule and initiates the transmission of a cell signal by binding to cross receptors, have previously been described e! a1., (1990) BVyuSyet, 265: 1449714504).

A number of data indicate the role of ΤΝΡ-α and ΤΝΡ-β as the main inflammatory cytokines. These well-known оказδ have an important physiological effect on the numerous and different target cells involved in the inflammatory response to various stimuli such as infection and damage. These proteins stimulate fibroblasts and synovial cells to secrete latent collagenase and prostaglandin E ₂ , and also stimulate osteocyte cells to resorb bones. These proteins increase the surface adhesive properties of endothelial cells with respect to neutrophils. They also induce the generation of coagulating activity by endothelial cells and reduce their ability to lyse blood clots. In addition, they redirect the activity of adipocytes from lipid storage by suppressing expression of the lipoprotein lipase enzyme. ΤΝΡδ induce the synthesis of such a class of proteins in hepatocytes that are known as acute phase reactants and which act on the hypothalamus as pyrogens (8е1Ьу е1 а1., (1988), Лапсе1 1 (8583): 483: 8!

1g e! A1., (1988), BS1t. 1way, 82: 1321; Οΐίίί e! A1., (1987), Ce11. 50: 555; \\ aade e! A1., (1987), Bayse !, 1 (8529): 355). The results of preclinical trials conducted on various models of inflammation in animals, including the model of rheumatoid arthritis, give reason to consider the suppression of ΤΝΡ as one of the main components of the progression of the disease and its severity (Eaueg e! A1., (1994), Eigoreap Su! Okte No. 1 \\ όγΕ. 5 (6): 563-571 and ReItapp e! a1., (1995), Lppak Οί Τίκ No. \ ν Ugk Lsabetu o! 8s1epsez, 66: 272-278). In addition, recent preliminary clinical trials of ΤΝΡ inhibitors in patients with rheumatoid arthritis have yielded encouraging results (Rapksh e! A1., (1995), Vyiky 1oyp1 o! Kiyeita! O1odu, 3 (4): 4334-4342: ЕШО! Е! А1. , (1995), Bapse, 344: 11051110: Also e! A1., (1996), ΛιΊΐιπΙίδ apb KBeitaSt, 29: 1077-1081; Raleo1od e! A1., (1996), ΛιΊΐιπΙίδ apb KBeitaSt, 39: 1082- 1091).

Protein inhibitors ΤΝΡ are known in the art. EP 308378 describes a protein obtained from the urine of patients with fever, having орной inhibitory activity. Apparently, the effect of this protein is due to a competitive mechanism at the receptor level. The protein described in the application EP 308378 was isolated in a sufficiently pure form, which made it possible to characterize its конец-end. In this work, however, no DNA sequence is given and the inhibitor ΤΝΡ obtained by genetic engineering methods is not described.

ΤΝΡ inhibitors obtained by genetic engineering methods are also known in the art. For example, EP 393438 and EP 422339 show the amino acid and nucleotide sequences of a mature recombinant human 30kD inhibitor ΤΝΡ (also known as a p55 receptor and 8ΤΝΡΡ.-Ι), as well as a mature recombinant human 40kD inhibitor (also known as p75- receptor and as 8ΤΝΡΚ.-ΙΙ), as well as its modified forms, in particular fragments, functional derivatives and variants. EP 393438 and EP 422339 also describe methods for isolating genes encoding inhibitors, cloning a gene in an appropriate vector and cell type, and gene expression to produce inhibitors. The ability of a mature recombinant human 30 kD inhibitor ΤΝΡ and a mature recombinant human 40 kD inhibitor ΤΝΡ to suppress ΤΝΡ has been shown (EP 393438, EP 422339, PCT publication No. O 92/16221 and PCT publication No. O 95/34326).

δΤΝΡΡ-Ι and δΤΝΡΡ-ΙΙ are members of the superfamily of nerve growth factor / tumor necrosis factor receptors (ΝΟΡ / рецеп), which include the nerve growth factor receptor (ΝΟΡ), CE40 B cell antigen. 4-1BB, rat T-cell antigen ISS OX40, antigen

EAC and antigens SE27 and SE30 (8thI e! A1., (1990), 8aepse, 248: 1019-1023).

The most conservative feature characteristic of the entire group of these receptors present on the cell surface is a cysteine-rich extracellular ligand binding domain that can be divided into four repeating structures of approximately forty amino acids and which contains 4-6 cysteine residues in highly conserved positions (§16 e! a1., (1990), Kirga).

EP 393438 discloses a 40 kDa inhibitor ΤΝΕ Δ51 and a 40 kDa inhibitor ΤΝΕ Δ53, which are truncated versions of the full length recombinant 40 kDa protein inhibitor ΤΝΕ at 51 or 53 amino acid residues, respectively, and the carboxy terminus of the mature protein is removed. Accordingly, it becomes clear to a qualified person that the fourth domain of both a 30 kDa inhibitor ΤΝΕ and a 40 kDa inhibitor is not necessary for inhibiting ΤΝΕ. In fact, this position has been confirmed by various groups of researchers. Derivatives of 30 kDa and 40 kDa of inhibitors of ΤΝΡ with domain divisions were obtained, while derivatives lacking the fourth domain retained full ΤΝΡ binding activity, derivatives without the first, second or third domain, respectively, did not retain ΤΝΡ-binding activity (Sogsogap e! A1., ( 1994) Eig.Wusziet., 223: 831-840: Sy-Nkiep e! A1., (1995), 1Ee 1oigpa1o £ B1o1od1ca1 SNGu. 270 (6): 2874-2878; 8ca11op e! A1., (1995) , Su! Okte, 7 (8): 759-770).

Due to the relatively low inhibition of cytotoxicity exhibited by a 30 kDa inhibitor ΤΝΡ and a 40 kDa inhibitor ΤΝΡ (Vi! 1eg e! A1., (1994), Cyukshe, 6 (6): 616-623), various research groups obtained dimers of protein inhibitors ΤΝΡ (ViNeg e! a1., (1994), Kirga; and Magip e! a1., (1995), Exp. Eigo1., 131: 221-228). However, dimers can elicit an antibody response (Magyp e! A1., (1995), Kirga; Iksyeg e! A1., (1996), Τίκ Iete Epdapi 1oigpa1 o £ Meiksha, 334 (26): 1697-1702).

The present invention is the creation of functionally active truncated κΤΝΡΚ, κ. This and other objectives of the present invention will be apparent from the description below.

SUMMARY OF THE INVENTION

The present invention is directed to the preparation of functionally active truncated forms κΤΝΡΒ-Ι and κΤΝΡΒ-ΙΙ, respectively, which are referred to as truncated κΤΝΡΒ (κ). Truncated κΤΝΡΚ, κ are modified forms of κΤΝΡΒ-Ι and κΤΝΡΒ-ΙΙ that do not contain the fourth domain (amino acid residues ΤΙη ^ -Άκο ¹⁶¹ κΤΝΡΒ-Ι and amino acid residues Рgo ¹⁴¹ -Tig ¹⁷⁹ κΤΝΡΒ-ΙΙ);

parts of the third domain (amino acid residues LKP ^1P- Suk ¹²⁶ κΤΝΡΒ-Ι and amino acid residues Рgo ¹²³ -Кук ¹⁴⁰ κΤΝΡΒ-ΙΙ); and, in addition, portions of the first domain (amino acid residues LCR'-Suk ¹⁹ 'κΤΝΡΒ-Ι and amino acid residues Leya ¹ - Suk ³² κΤΝΡΒ-ΙΙ).

These new ΤΝΡ inhibitors (in particular, ΤΝΕα and ΤΝΡβ) have wide applicability.

According to the present invention, truncated κΤΝΡΚ, κ include proteins represented by the formula B1- [Suk ^19- Suk ¹⁰³ ] -B2 and B4- [Suk ³² Suk ¹¹⁵ ] -B5. These proteins are truncated forms of κΤΝΡΒ-Ι and κΤΝΡΒ-ΙΙ, respectively.

Formula B ₁ - [Suk ^19- Suk ¹⁰³ ] -B ₂ 'denotes one or more proteins in which [Suk ^19- Suk ¹⁰³ ] represents residues from 19 to 103 κΤΝΡΒ-Ι, and the numbering scheme of amino acid residues is used to facilitate comparison shown in FIG. 1 (8ETS ΙΌ ΝΟ: 2), and B! denotes a methionylated and nonmethionylated 19 amino group Suk or an amino terminal amino acid residue selected from group c

1C

31C

Ν3ΙΟ (ZEO GO N0: 15)

ΝΝ5ΙΟ (ZEO GO N0: 16)

ΟΝΝ5ΙΟ (ZEO GO N0: 17)

ΡΟΝΝ5ΙΟ (ZEO GO N0: 18)

ΗΡΟΝΝ3ΙΟ (ZEO GO N0: 19)

ΙΗΡΟΝΝ5ΙΟ (ZEO GO N0: 20)

ΥΙΗΡΟΝΝ3ΙΟ (ZEO GO N0: 21)

ΚΥΙΗΡΟΝΝ3ΙΟ (ZEO GO N0: 22)

ΟΚΥΙΗΡ0ΝΝ5ΙΟ (ZEO GO N0: 23)

ΟΟΚΥΙΗΡΟΝΝ3ΙΟ (ZEO GO N0: 24) ΡΟΟΚΥΙΗΡΟΝΝ3ΙΟ (ZEO GO N0: 25) ΟΡΟΟΚΥΙΗΡΟΝΝΒΙΟ (ZEO GO N0: 27) 5νθΡΟΟΚΥΙΗΡΟΝΝ3ΙΟ (ZEO GO N0: 28) ϋ3νθΡΟΟΚΥΙΗΡΟΝΝ5ΙΟ (ZEO GO N0: 29), and In ₂ denotes a carboxy group Suk ¹⁰³ or carboxy-terminal amino acid residue selected from group g

ΕΝ

ΡΝΟ

ΡΝ08 (ZEO GO N0: 30)

ΡΝ08Ε (ZEO GO N0: 31)

ΡΝ03Ε0 (ZEO GO N0: 32)

ΡΝ08Ε0ί (ZEO GO N0: 33), as well as their variants and derivatives, obtained as described in the description, included in the scope of the invention, when B ₁ denotes a methionylated or non-methionylated amino group in the amino acid sequence νί'ίδΙί 'or its Ν-terminal truncated by 1-15 residues of the variant, despite the fact that the protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K2 is not an additional variant with the formula Y1- [Su8 ¹⁹ -Su8 ¹⁰³ ] -EMC8Sb-K3, and where K ₃ represents a carboxyl group amino acid sequence LyS-Ly! ¹⁶¹ . shown in FIG. 1, or a carboxy-terminal truncated variant thereof.

Examples of truncated δΤΝΕΚ-Ι related to the present invention include the following molecules:

\ H; - \ 11) 8 \ G1CH) 1 # P 114) 448 ((4 (^ Suk ¹⁰⁵ ] -EC-COOH (also denoted as δΤΝΡΚ-Ι 2.6P / C105);

4'11 _; - \ 11) 8 \ 'CP () KX'P 114) 448, (4 (8 ^ Su5 ^{| 05} | -SLB-COOH (also denoted as δΤΝΡΚ-Ι 2.6O / C106);

4'11 _; - \ 11) 8 \ 'C14) KH'P 114) 44844 () ^ Suk ¹⁰⁵ ] -ΕΝ-COOH (also denoted as δΤΝΕΚ-Ι 2.6Ό / Ν105);

1MN _2- M ¥ SHRr] \ 1 # 1C- [Su8 ¹⁹ -Cu8 ¹⁰⁵ ] E4C8E-COOH (also denoted as δΤΝΕΚ-Ι 2.3Ό / 68);

1MH2-M- [Su8 ¹⁹ -Su8 ¹⁰⁵ ] -E4C8P-COOH (also denoted as δΤΝΕΚ-Ι 2.3Ό / 618);

NH2-M8I8- [Suk ^19- Suk ¹⁰⁵ ] -NC8 ^ -COOH (also denoted as δΤΝΕΚ-Ι 2.3Ό / 615), as well as their methionylated and non-methionylated forms, their variants and derivatives.

Thus, the formula K ₄ - [Suk ^32- Suk ¹¹⁵ ] K5 denotes one or more proteins in which [Suk-Suk] represents residues from Suk to Suk ¹¹⁵ κΤΝΕΚ-Ι, and the numbering scheme of amino acid residues is used to facilitate comparison shown in FIG. 8 (8EO ΙΌ NO: 35), and K4 represents the methionylated and non-methionylated amino group Suk ³² or amino-terminal amino acid residues selected from group c

MS

Ome

AOMS (KEO GO N0: 36)

TA0MS (KEO GO N0: 37)

OTAOMS (KEO GO N0: 38)

OOTAOMS (KEO GO N0: 39)

UOOTAOMS (KEO GO N0: 40)

UUOOTAOMS (KEO GO N0: 41)

ΕΥΥϋΟΤΑΟΜΟ (KEO GO N0: 42) ΚΕΥΥΟΟΤΑΟΜΟ (KEO GO N0: 43) KKEUUOOTAOOMS (KEO GO N0: 44) KKEUOOOOTAOMS (KEO GO N0: 45) SKEUUOOTAOMS (KEO GO N0: 46) TSKVEUUOOOTOMSOT (47) KEO GO N0: 48)

OKTSKKEUUOOOOTAOMS (KEO GO N0: 49)

ROKTSVKEUUOOOOTAOOMS (KEO GO N0: 50)

EROKTSKKEUUOOOOTAOOMS (KEO GO N0: 51)

REROKTSKEUTOOTOAOMS (KEO GO N0: 52)

AREROKTSVKEUUOOOOTAOMS (KEO GO N0: 53)

UAREROZTSKKEUTOOTLOMS (8E0 GO N0: 54) RUARERSZTSKKEUUOOTAOMS (Zec GO N0: 55) TRUAREROZTSKKEUUOOTAOMS (5E0 GO N0: 56) ETRUARERO5TSM.KEUUOTSTA0MS (5E0 GO N0: 57) AETRUAREROZTSVVEUUOOTATSMS (5E0 GO N0: 58) UAETRUAREROZTSKKEUUOTSTAOMS (5E0 GO N0: 59) 0UAETRUARERO5TSKKEUUO0TA0MS (WE <2 GO N0: 60) A0UAETRUARERO5TSKEKEUUP <irA <} MS (ZEO GO N0: 61) RAOUAETRUAREROZTSKVEGUOOTAOMS (ZEO GO N0: 62) RAOUAETRUARER05TSK1LEUUP <2TAOMS (ZEO GO N0: 63) and K ₅ is carboxy Suk ¹¹⁵ or carboxyterminal amino acid residue (s) selected from the group

BUT

AR

AR

ARK (KEO W N0: 64).

ARKK (KEO GO N0: 65).

ARKKS (KEO GO N0: 66).

ARKKSK (KEO GO N0: 67), as well as their variants included in the scope of the invention, when K ₄ denotes a methionylated or non-methionylated amino group in the amino acid sequence

Τ ^ ΡΡΕΥΥΟΟΤΛΟΙ ^. ' or its Ν-terminal truncated by 1-15 residues variant while the protein K ₄ - [Suk ³² - Suk ¹¹⁵ ] -K5 is not an additional option with the formula K4 [Suk ³² - Suk ¹¹⁵ ] -LRKKSK.-K6, and where Kb is the carboxyl group of the amino acid sequence Pro-Τίπ shown in FIG. 8, or a carboxyterminally truncated variant thereof.

According to one aspect of the present invention, truncated κΤNΕKκ can be obtained in glycosylated or non-glycosylated forms. Truncated κΤNΕКκ are obtained by genetic engineering methods. In an alternative embodiment of the invention, truncated ^ ΝΕΡκ can be obtained by chemical synthesis methods or by a combination of chemical synthesis methods and genetic engineering methods.

According to another aspect of the present invention, truncated κΤNΕK.κ can be derivatized by attaching a water-soluble polymer to the truncated ^ ΝΕΡκ. For example, truncated ^ ΝΕΡκ can be conjugated to one or more polyethylene glycol molecules in order to improve pharmacokinetic performance by increasing the effective molecular weight of the molecule.

Another aspect of the present invention are various polynucleotides encoding truncated κΤNΕKκ. Acceptable nucleotide sequences include those shown in the drawings, as well as degenerate sequences and naturally occurring allelic variants. These nucleotide and amino acid sequences can be used to express truncated EXTs in eukaryotic and prokaryotic host cells, and the expression product or its derivative is characterized by the ability to modulate ΤΝΡ activity.

Another aspect of the present invention includes vectors containing coding for truncated δΤΝΡΚδ polynucleotides operably linked to sequences that provide amplification and / or control expression. For the expression of truncated δ такимиδ by such vectors, both prokaryotic and eukaryotic host cells can be transformed or transfected. Methods for the recombinant production of truncated δΤΝΡΚδ are also included within the scope of the invention, while host cells containing such polynucleotides are grown on an acceptable nutrient medium and truncated δΤΝΡΚδ expressed by cells are isolated from the host cells and / or from the nutrient medium.

Another aspect of the present invention are pharmaceutical compositions comprising truncated δΤΝΡΚδ or derivatives thereof. Typically, truncated δΤΝΡΚδ or derivatives thereof are formulated in combination with pharmaceutically acceptable carriers. To facilitate the production, storage, handling, delivery and / or increase the efficiency of truncated δΤΝΡΚδ or their derivatives, various other materials may be included in the compositions.

Another aspect of the present invention relates to methods for modulating ΤΝΡ activity. In particular, ΤΝΡ-caused diseases (for example, diseases caused by ΤΝΡ-α and / or ΤΝΡ-β) can be treated by administering to the patient therapeutically effective amounts of truncated δΤΝΡΚδ or their derivatives.

Polynucleotides encoding truncated δΤΝΡΚδ can also be used for cell or gene therapy.

The truncated δΤΝΡΚδ obtained according to the present invention are particularly suitable for producing large quantities of protein. For example, δΤΝΡΚ-Ι in amino acid sequence 111-126 (amino acids Λδη ^1Π -Ο ^) ¹²⁶ has one deamination site. It is believed that the absence of this site should enhance the biochemical stability of the purified protein, reducing the number of possible degradation products and increasing the stability of proteins during storage. Truncated δΤΝΡΚδ have fewer disulfide bridges than the previously described ΤΝΡ inhibitor proteins. For example, δΤΝΡΚ-Ι contains two disulfide bridges in the amino acid sequence 111-126 and three disulfide bridges in the amino acid sequence 127-161; δΤΝΡΚ-Ι Ι contains disulfide bridges between Οΐδ ¹²¹ and Οίδ ¹³⁹ , Οΐδ ¹⁴² and Οΐδ ¹⁵⁷ , Οΐδ ¹⁶³ and Οϊδ ¹⁷⁸ . A reduced number of disulfide bridges is important because a greater number of such bonds can complicate the process of protein rearrangement. Surprisingly, truncated δΤΝΡΚδ have fewer sites of antigenic epitopes than previously described protein inhibitors ΤΝΡ (for example, the truncated form of δΤΝΡΚ-Ι containing the first three domains has neoepitopes whose appearance is due to exposure of individual residues, see Example ΙΙΙ), which leads to a comparative reduced antigenicity, but does not significantly reduce the clearance rate during repeated injections. The reduced immunogenicity of truncated δΤΝΕΚ, δ, as expected, can be a positive property in the treatment of ΤΝΡ-caused diseases, in particular chronic inflammatory diseases.

Additional aspects and advantages of the present invention will become apparent to those skilled in the art upon consideration of the following description, which details the application of the present invention.

Brief Description of the Drawings

Numerous aspects and advantages of the present invention will become apparent upon analysis of the following drawings.

In FIG. 1 shows the nucleotide sequence (8EO ΙΌ ΝΟ: 1) encoding Λδρ'-Λδη ^{161 of a} full-sized recombinant δΤΝΡΚ-Ι person. The amino acid sequence (BEO ΙΌ ΝΟ: 2) Λδρ'Λδη ^{161 is also given} .

In FIG. Figure 2 shows the nucleotide sequence (BEO ΙΌ ΝΟ: 3) encoding ΝΗ ₂ -ΜΌ8νΕΡΟΚΥΙΗΡΟΝΝ8ΙΕ- | Ενδ ^Ι9 -Ενδ ^Ιυ5 | KC-COOH (also denoted as δΤΝΡΚ-Ι 2.6Ό / ί.Ί05). The amino acid sequence is also given (BEO ΙΌ ΝΟ: 4) NAN ₂ mp ^ srststrrmms ^ ¹⁹ ^ ¹⁰⁵ ] ^ (ΌΟΙΙ.

In FIG. 3 shows the nucleotide sequence (BEO ΙΌ ΝΟ: 5) encoding ΝΗ _; -ΜΌ8νί: ΡΟΙ <ΥΙΗΡΟΝΝ8Κ: - | ί \ δ ¹⁹ -ί \ δ ^1ο5 | ΡNC8 ^ -COOH (also denoted as δΤΝΡΚ-Ι 2.6Ό / Ο06). The amino acid sequence (BEO ΙΌ ΝΟ: 6) NН ₂ -Μ ^ 8VСΡ ^ ΚΥINΡ ^ NN8IС- [Suδ ¹⁹ -СУδ ¹⁰⁵ ] ΡΝ ^ 8Ό <ΟΟΗ is also given.

In FIG. 4 shows the nucleotide sequence (BEO ΙΌ ΝΟ: 7) encoding ΝΗ _; -ΜΌ8νί: ΡΟΙ <ΥΙΗΡΟΝΝ8Κ: - | ί \ δ ¹⁹ -ί \ δ ^1ο5 | ΡΝ ^ ΟΟΗ (also denoted as δΤΝΡΚ-Ι 2.6Ό / Ν105). The amino acid sequence (BEO ΙΌ ΝΟ: 8) YN ₂ Μ ^ 8VСΡ ^ ΚΥIΗΡ ^ NN8IС- [Suδ ¹⁹ -Suδ ¹⁰⁵ ] -N (ΌΟΙΙ.

In FIG. 5 shows the nucleotide sequence (BEO ΙΌ ΝΟ: 11) encoding ΧΙΕ-ΜΥΙΗΡΟΧΝΛΚ'-Ιίλδ-ίλδΊ-ΡΧί '^ Ρί,' ΟΟΗ (also denoted as δΤΝΡΚ-Ι 2.3Ό / 68). The amino acid sequence (BEO ΙΌ ΝΟ: 12) ΝΗ ₂ ΜΥΙΗΡΟΧΧΛΚ'-Ιίλδ-ίλδΊ-ΡΧίΧΡ-ί'ΟΟΙΙ is also given.

In FIG. Figure 6 shows the nucleotide sequence (8EO ΙΌ N0: 9) encoding No. ₂ -M- [Cy8 ¹⁹ -Cu8 ¹⁰⁵ ] -E ^ 8L-COOH (also denoted as δΤΝΕΕ-Ι 2.3Ό / ά18). The amino acid sequence (8 HO ΙΌ N0: 10) \ 'H ₂ - \ 1- | C \ 5 -C \ X | -E \' C8ES00H is also given.

In FIG. 7 shows the nucleotide sequence (8E0 ΙΌ N0: 13) encoding ^ 2-M818- [Cy5 ¹⁹ -Cu8 ¹⁰⁵ ] -E ^ 8E-COOH (also denoted as δΤΝΕΕ-Ι 2.3Ό / 615). The amino acid sequence (8 BUT ΙΌ ^^ Ing-MBK-GSu ^ -Souss ¹⁰⁵ ] ENC8B-C00H is also given.

In FIG. Figure 8 shows the nucleotide sequence (8E0 ΙΌ N0: 34) encoding Ley ¹ -Tm ¹⁷⁹ mature recombinant human kTNEV-E. The amino acid sequence (8E0 ΙΌ N0: 35) of Le ¹ -Tm ^{179 is also given} .

In FIG. 9 presents data on measuring the degree of edema induced when using the arthritis reactivation model using streptococcal cell wall products, as described in Example ΙΙ.

In FIG. 10 presents plasma profiles xTNE-! 4E / C105 in healthy baboons after a two-minute intravenous administration of 0.2 mg / kg, as described in example ΙΙΙ.

In FIG. 11 presents plasma profiles xTNE-! 3E / C105bb in healthy baboons after a two-minute intravenous administration of 0.2 mg / kg, as described in example ΙΙΙ.

In FIG. 12 shows the plasma profiles of CTOTKN 2.6E / C105b in healthy baboons after a two-minute intravenous administration of 0.2 mg / kg, as described in example ΙΙΙ.

In FIG. 13 shows the relationship between dose and clearance for various dimeric constructs of CTOTCN, as described in Example ΙΙΙ.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected observation that 5THEE- [and kT№K.ΙΙ can be reduced in size by removing not only the fourth domain, but also part of the third domain and, in addition, part of the first domain, while maintaining biological activity and having a reduced antigenicity. At least for the following reasons, the preparation of said biologically active truncated kT # K.k. or derivatives thereof may present certain advantages. First, these molecules have one less potential destabilizing deamination site. Secondly, these molecules contain less disulfide bridges, which potentially facilitates re-laying and cleaning. Thirdly, these molecules have a reduced number of sites of potential antigenic epitopes.

The term truncated kT№K. (K) combines one or more biologically active synthetic or recombinant molecules with the formula B ₁ - [Suk ^19- Suk ¹⁰³ ] -B2 or B4- [Suk ³² Suk ¹¹⁵ ] -K.5, as well as their variants (including insertion variants, variants with substitutions and deletions), as described below.

The term biologically active means that truncated kT№K.k have similar T№-inhibitory properties, but not necessarily the same properties and not necessarily to the same extent as kyutkn and / or kTNK.-P. Generally speaking, truncated kT№K.k and their derivatives are able to inhibit UT. Biological tests for the activity of truncated kT№K.k are described further in Example ΙΙ. The choice of specific UT-inhibitory properties that are of interest depends on the method of application of truncated UTCB.

According to one aspect of the present invention, truncated cTKK.c is preferably prepared using recombinant techniques in bacterial, mammalian or insect cell systems, while truncated kTKK can be both glycosylated and non-glycosylated forms of the protein. Alternatively, truncated cuTBC may be chemically synthesized. The currently most preferred production methods are described in more detail below.

Truncated kTKEK.k can be isolated and purified in a form substantially free of the presence of another (i.e., not truncated kTKK.k) protein material. Preferably, the truncated kT # K.k. is 80% free of other proteins that may be present in the preparations by virtue of the method used to produce the truncated kT # K. More preferably, truncated kT # K.k is 90% free of other proteins, particularly preferably 95% free of other proteins, and most preferably more than 98% free of other proteins. However, it is understood that the desired protein can be combined with other active ingredients, chemical compositions and / or acceptable pharmaceutical formulations before administration, as described in more detail below.

Truncated kT№K.k.

According to one of the main embodiments of the present invention, truncated cJTBKs include proteins represented by the formula B ₁ - [Suk ^19- Suk ¹⁰³ ] -B2, in which [Suk ^19- Suk ¹⁰³ ] represents residues from 19 to 103 kTOTKN, while for to facilitate comparison, the numbering scheme of amino acid residues shown in FIG. 1 (8E0 ΙΌ N0: 2), and K.1 denotes the methionylated and non-methionylated amino group of Suk or the amino terminal amino acid residues selected from group c

1C Zu N310 (ZEO GO N0: 15) Nn510 (5E0 GO N0: 16) 0ΝΝ3ΙΟ (5E <E GO N0: 17) Ρ0ΝΝ5Ι0 (5E <2 No.N0: 18) ΗΡ0ΝΝ5ΙΟ (5E0 GO N0: 19) ΙΗΡ0ΝΝ5Ι0 (5E0 GO N0: 20) ΥΙΗΡ0ΝΝ5ΙΟ (5E0 GO N0: 21) Ι <ΥΙΗΡζ) ΝΝ8ΙΟ (ZEO GO N0: 22)

ΟΚΥΙΗΡ0ΝΝ5ΙΟ (Zeo Go N0: 23) ΟΟΚΥΙΗΡζ) ΝΝ5ΙΟ (ZEO GO N0: 24) ΡΟΟΚΥΙΗΡΟΝΝ8ΙΟ (ZEO GO N0: 25)

ΟΡΟΟΚΥΙΗΡΟΝΝ5ΙΟ (ZEO GO N0: 26) νΟΡΟΟΚΥΙΗΡΟΝΝΒΙΟ (3Εζ) GO N0: 27)

8νθΡΟΟΚΥΙΗΡΟΝΝ5ΙΟ (ЗЕ <Э ГО N0: 28) ϋ3ν0Ρ <26ΚΥΙΗΡ0ΝΝ5Ι0 (5Εζ) ГО N0: 29), and K ₂ denotes the carboxy group of СУЗ ¹⁰³ or carboxyterminal amino acid residues selected from the group

E

ΡΝ LYS ΡΝ08 (ZEO GO N0: 30) Lynx (5E <3 GO N0: 31) RYSZS (ZEO GO N0: 32) ΡΝΟδΙΧΧ (ZEO GO N0: 33),

and their variants and derivatives, obtained as disclosed in the description, included in the scope of the invention, when K ₁ denotes a methionylated or non-methionylated amino group in the amino acid sequence νί, 'ΡΟΟΚΥΙΗΡΟΝΝίίΙί.' or its Ν-terminal truncated by 1-15 residues variant despite the fact that the protein K ₁ - [Suz ^19- Suz ¹⁰³ ] -K2 is not an additional option with the formula K1- [Suz ¹⁹ Suz ¹⁰³ ] -P ^ 8Sb-K3, and where K ₃ denotes the carboxyl group of the amino acid sequence of Alp ¹¹¹ -Azp ¹⁶¹ shown in FIG. 1, or a carboxy-terminal truncated variant thereof.

According to another main embodiment of the present invention, the truncated z'T # K3 can be proteins denoted by the following formula: I.- | - | C.'Uz ³² Suz] -K5, in which [Suz-Suz] represents residues from Suz ³² to Suz ¹¹⁵ zT№K.-P, while for ease of comparison, the numbering scheme of amino acid residues shown in FIG. 8 (8 EC ΙΩ N0: 35), and K4 represents the methionylated and non-methionylated amino group Suz ³² or amino-terminal amino acid residues selected from group c

MS <2MS

AfMS (5Εζ) GO N0: 36)

TAfMS (5E <2 GO N0: 37)

0TA0MS (5Е0 ΙΟ N0: 38) EOTAOMS (5Εζ> ГО N0: 39) ΥΟζ> ΤΑζ> ΜΟ (5Е <2 ГО N0: 40) УГООТААМС (ЗЕО ГО N0: 41)

ΕΥΥϋΟΤΑΟΜΟ (5Εζ) GO N0: 42) ΚΕΥΥϋΟΤΑΟΜΟ (5Е0 GO N0: 43) LKEUUOOTAOMS (5Е0 GO N0: 44) M. KEUUO0TA0MS (5Εζ) GO N0: 45) SKKEUUOOTAOMS (8Е0 GO N0: 46) TSCKEUUOOOTAOMS : 47) ZTSKKEUUIOTAOMS (5Е0 ГО N0: 48) ОЗТСКЬКУУУУУОТАУСОС (5Е0 ГО N0: 49) ROZTSKKEUUOOTAOMS (5Е <Э GO N0: 50) ЕРОЗТЬСЬЕУУУОУОСОТ (5Εζ> ГООЗЕЗОЗОЗОЗОЗО5: 5 GO N0: 53) UAREROZTSKKEUUPOTAOMS (5E "E GO N0: 54) RUAREROZTSKKEUUPOTAOMS (5Εζ> GO N0: 55) TRUARERSZTSKKEUUOOTAOMS (5Εζ) GO N0: 56) GTRUARERSETSK1.KEUU0 <ETA0MS (ZEO GO N0: 57) ARTRUARERSETSKKEUUOOTAOMS (5Εζ) GO N0: 58)

UAETRUAREROZTSKKEUUPOTAOMS (8E <GO N0:? 59) OUAGTRUAREROZTSKKEUUPOTAOMS (8E0 GO N0: 60) AOUAETRUAREROZTSKKEUUOOTAOMS (5E0 GO N0: 61) RAOUASTRUARERSZTSKKEUUOOTAOMS (5E0 GO N0: 62) ERAOUAETRUAREROZTSKKEUUOOTAOMS (5E0 GO N0: 63) and K ₅ is carboxy Cys ¹¹⁵ or carboxyterminal amino acid residue (s) selected from the group

BUT

AR AR ARK (8Εζ) GO N0: 64) ARKK (8E0 GO N0: 65) ARKKS (5E0 U N0: 66) ARKKSV (8E0 GO N0: 67), and their variants, also included in the scope of the invention, when K ₄ denotes the methionylated or non-methionylated amino group in the amino acid sequence

Τ ^ ΡΡΕΥΥΟΡΤΑΟΙ ^ '.' or its Ν-terminal truncated by 1-15 residues variant, despite the fact that the protein K ₄ - [Suz ^32- Suz ¹¹⁵ ] -K5 is not an additional option with the formula K4 [Suz ^32- Suz ¹¹⁵ ] -ARKKSK-K6, and where K ₆ denotes the carboxyl group of the amino acid sequence Prgo-Tbm shown in FIG. 8, or a carboxyterminally truncated variant thereof.

Another aspect of the present invention relates to one or more variants of K ₁ - [Suz ^19- Suz ¹⁰³ ] -K2 and K4- [Suz ^32- Suz ¹¹⁵ ] -K5 in both methionylated and non-methionylated forms. Thus, the term truncated zT№K. (H) includes one or more natural allelic variants of K _g [Suz ^19- Suz ¹⁰³ ] K2 and K4- [Suz ^32- Suz ¹¹⁵ ] -K5 and one or more other protein variants, in which amino acids have been delegated (deletion variants), inserted (variants with additions) or replaced (variants with replacements) with residues in the amino acid sequence K ₁ [Suz ^19- Suz ¹⁰³ ] -K, 2 or K.4- [Suz ^32- Suz ¹¹⁵ ] -K_5.

Amino acid sequence deletions typically vary within 20 amino acid residues, more typically from 1 to 10 amino acid residues, and most typically from 1 to 5 residues so that protein folding is not disturbed. We consider Ν-terminal, C-terminal, and internal intrasequential deletions. The total number of deletions and / or consecutive deletions is chosen in such a way as to preserve the quaternary structure of the protein in the involved domain, i.e., cross-linking of cysteine.

Deletions in the amino acid sequence K ₁ - [Suk ^19- Suk ¹⁰³ ] -B2 and in the amino acid sequence K4- [Suk ^32- Suk ¹¹⁵ ] -K5 can be performed in regions of low homology with sequences of other members of the ΝΟΡ / торов receptor family in the surface group membrane protein cells. Deletions in the amino acid sequence K ₁ [Suk ^19- Suk ¹⁰³ ] -K2 and in the amino acid sequence K4- [Suk ^32- Suk ¹¹⁵ ] -K5 can be made in areas of significant homology with sequences of other members of the ΝΟΡ / торов receptor family, and in this case will more significantly modify biological activity. In particular, the sequence similarity of the членов / торов receptor family members is especially great in the region corresponding to the first two disulfide loops of domain 1, the entire domain 2, and the first disulfide loop of domain 3 (Wappeg c1 a1., (1993), Ce11, 73: 431-445 ) For example, two deletion variants K. ₁ - [Suk ^19- Suk ¹⁰³ ] -B2 are K1- [Suk ¹⁹ (ATyg ²⁰ ) -Suk ¹⁰³ ] -K.2 and K1 [Suk ¹⁹ (LSUK ^19- Suk ²¹ ) -SUK ¹⁰³ ] -K.2, where Kl and K2 are defined above. For example, three deletion variants of K4- [Suk ^32- Suk ¹¹⁵ ] -B5 are K ₄ [Suk ³² - (LSuk ¹¹⁵ ) -Suk ¹¹⁵ ] -K.5; K4- [Suk ³² - (ASuk ¹¹⁵ Luk ¹¹⁵ ) -Suk ¹⁰³ ] -K.5 and K.4- [Suk ³² - (LSuk ^115- Agd ¹¹³ ) Suk ^{|| 5} | -K5. where K ₄ and K _{5 are} defined above.

Additions to the amino acid sequence may include amino and / or carboxyterminal fusions ranging in length from one residue to hundreds or more residues, as well as internal intra-sequence insertions of single or multiple amino acid residues. Internal inserts typically vary in size from 1 to 10 amino acid residues, more typically from 1 to 5 amino acid residues, and most typically from 1 to 3 amino acid residues.

Options with amino-terminal additions include the addition of methionine (for example, to ensure direct expression of the protein in a recombinant bacterial cell culture) or the addition of an additional amino acid residue or sequence. Another example of an amino terminal insertion is fusion with a signal sequence, as well as with other pre-sequences, to facilitate the secretion of protein from recombinant host cells. For prokaryotic host cells that do not recognize or process native κΤΝΡΒI or κΤΝΡΒ-сигна signal sequences, these signal sequences can be replaced with prokaryotic signal sequences selected, for example, from the group comprising leader sequences of alkaline phosphatase, penicillinase or thermostable enterotoxin II. For yeast cells, the signal sequence can be selected, for example, from the group comprising the leader sequences of yeast invertase, alpha factor or alkaline phosphatase. For expression in mammalian cells, the native κΤΝΡΒ-Ι or κΤΝΡΒ-сигна signal sequences (EP 393 438 and EP 422 339) are quite acceptable, although other mammalian signal sequences (for example, sequences obtained from other members of the ΝΟΡ / ΤΝΡ receptor family may be convenient) )

Options with carboxyterminal additions do not include the addition of one or more amino acid residues, which would lead to the reconstruction of κΤΝΡΒΙ or κΤΝΡΒ-ΙΙ, respectively. It is understood that variants with carboxyterminal additions do not include the addition of one or more carboxylic acid residues, which would lead to the reconstruction of the third domain or the fourth domain κΤΝΡΚ-Ι or κΤΝΡΒ-ΙΙ. Examples of carboxy-terminated variants include chimeric proteins resulting from the fusion of ^ - [Suk ¹⁹ Suk ¹⁰³ ] -K2 or B4- [Suk ^32- Suk ¹¹⁵ ] -B5 with the full or partial constant domain of a human immunoglobulin heavy or light chain. Such chimeric proteins are preferred because the immunoglobulin portion represents all domains except the first domain of the constant region of the heavy chain of human immunoglobulins, such as Ι§Ο, Ι§Α, Ι§Μ or Ι§Ε, especially Ι§Ο, i.e. Ι§Ο2 or Ι§Ο3. It is obvious to a qualified person that any amino acid of each immunoglobulin moiety can be deleted or replaced with another or other amino acids, one or more amino acids can be added, while the ΤΝΡ-binding moiety retains the ability to bind ΤΝΡ, and the immunoglobulin moiety retains one or more of its characteristic properties .

Another group of options are variants with amino acid substitutions. In the case of these options, at least one amino acid residue K ₁ - [Suk ^19- Suk ¹⁰³ ] -K. or K2 [Suk ^32- Suk ¹¹⁵ ] -B4 is removed and another residue is inserted in its place. Substitutional variants include allelic variants characterized by natural substitutions in the nucleotide sequence of a population of a given species, which may or may not lead to amino acid substitutions. A qualified specialist can use any information available about the binding or active site of the polypeptide when selecting possible mutation sites.

One of the methods for identifying amino acid residues or regions for protein mutagenesis is called alanine-scanning mutagenesis (Sipspdyat apb ^ e11k (1989), Bc1epse, 244: 1081-1085, the work is mentioned in the present description by reference). In this approach, an amino acid residue or a group of target residues is identified (for example, charged residues such as Agd, Akr, Shk, Luk, O1u) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to change the interaction of amino acids with the surrounding aqueous environment inside or outside the cell. Such residues exhibiting functional sensitivity to substitutions are then scrubbed by introducing additional or alternative residues at the substitution sites. Thus, the site for introducing the modification into the amino acid sequence is predetermined and, to optimize the mutation, alanine or random mutagenesis is scanned at this site, and the resulting polypeptide is screened for the optimal combination of the desired activity and the degree of this activity.

Sites of particular interest for substitutional mutagenesis include those sites that are found in K. ₁ [Suk ^19- Suk ¹⁰³ ] -K.2 and 1T- | C \ k ^;; -C \ k '| -H, amino acids differ significantly in the presence of side chains, charge and / or hydrophobicity from κΤΝΡΚ-like proteins, such as κΤΝΡΚκ of other biological species or other members of the Ν0Ρ / ΤΝΡ receptor family.

Sites in which specific residues are identical to those in κΤΝΡΚ-1-like proteins and in κΤΝΓΈ-ΙΙ-like proteins are also of interest. These provisions are usually important for maintaining the biological activity of the protein. For example, one skilled in the art will understand that prior to the present invention, the effects of truncation κΤΝΡΚ-Ι and κΤΝΕΚ-ΙΙ on their three-dimensional structures were unpredictable. However, taking into account the results presented in this description, a qualified specialist can understand that the principles of developing a strategy for obtaining options can be partly determined by the information obtained earlier for full-sized κΤΝΓΉ-Ι and κΤΝΡΚ-ΙΙ. Accordingly, similar information regarding κΤΝΓΉ-Ι was published (Wappeg e! A1., (1993), Kirga and Rts e! A1., (1995), Proc. Residues Tug ⁹ , ΤΡγ ¹⁹ , Ηίκ ⁵⁵ in domain 1, residues Rye ⁴⁹ , Run ⁶³ ,

Acre ⁸² in domain 2 and the remains of Tug ⁹² , Running ⁶³ in domain 3 were identified as potentially important for stabilizing the structure of domains 1, 2, and 3, respectively. Residues of Rgo ¹² and Ηίκ ⁵⁵ were identified as potentially interacting with Beg ⁸⁶ -Tug ⁸⁷ subunits of C ΤΝΡα. Residues O1c ⁴⁵ -Pye ⁴⁹ were identified as forming a loop that potentially interacts with the residues Lea ^29- Agd ³² subunits A ΤΝΡ α. Residue O1y ⁴⁸ was identified as potentially interacting with Akp ¹⁹ –Rgo ²⁰ subunit A ΤΝΡα. Residues Shk + Ley ⁶⁰ were identified as forming an extended chain conformation; In addition, the potential interaction of the side chains of the Agd ³¹ -A1a ³³ residues of the A ΤΝΡα subunit with the H1k residue of ⁵⁸ κΤΝΡΡ-Ι was shown. which specifically interacts with Agd ³¹ residue. Residues Luc ⁶⁴ -Agd ⁶⁶ were identified as forming an extended chain conformation; in addition, the potential interaction of the side chains and the main chains of these residues with residues A1a ¹⁴⁵ -O1c ¹⁴⁶ and residue 01c ^{46 of} subunit A ΤΝΡα was shown. The rest of Me! ⁶⁹ was identified as potentially interacting with the residue of Tug ¹¹⁵ subunit A ΤΝΡα. Residues Ηίκ ⁹⁴ PHe ¹⁰¹ were identified as forming a loop that interacts with the residues Pn ^ Rey ⁵ and Akp ^{137 of} subunit A ΤΝΡα; the residue Tgr ⁹⁶ κΤΝΡΚ-Ι specifically interacts with the residues Be ^ ⁷¹ -th ^ ⁷² subunits of C Сα; the Lea ¹⁰⁰ κΤΝΡΚ-Ι residue is in close proximity to the Akp residue ^{137 of} the C ΤΝΡ α subunit, and the O1n residue ¹⁰² κΤΝΡΚ-Ι specifically interacts with the Prgo residue ^{113 of} the A ΤΝΡα subunit. Accordingly, it is obvious to a qualified person that initially these sites are modified by substitutions introduced in a relatively conservative way.

Such conservative substitutions are given in table. 1 under Preferred Substitutions. If such substitutions lead to a change in biological activity, then more substantial substitutions are made (Possible substitutions). Other additions or deletions can also be made, followed by screening for the resulting polypeptides.

Table 1 Amino Acid Substitutions

Original balance Preferred replacements Possible replacements A1a (A) Ya1 Wa1; Lei; Not; Agd® Yuk Luke; O1p; Akp Akp (Ν) O1p O1p; Shk; Luke; Agd Acre (E) O1i O1i Bitch © Run Run O1p (0) Akp Akp O1i (E) Acre Acre O1y (O) Rgo Rgo Ηίκ (Η) Agd Akp; O1p; Luke; Agd

11e (I) Bie Lei; Vaί; Me !; A1a; RBE; norleucine Bie (b) 11th norleucine; 11e; Vaί; Me !; A1a; RBE Luke (C) Agd Agd; O1p; Akp Me! (M) Bie Lei; RBE; 11th RBE (G) Bie Lei; Vaί; 11e; A1a Rgo (R) O1u O1u 8eg (8) Tbg Tht Tbg (Τ) 8eg 8eg Tgr (U) Tug Tug Va1 (V) Bie 11e; Lei; Me !; RBE; A1a; norleucine

When making such replacements for equivalents, the hydropathic index of amino acids must be taken into account. Each amino acid has its own hydropathic index, determined on the basis of its hydrophobicity and charge characteristics: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

The importance of the hydropathic amino acid index in maintaining the biological function of a protein associated with its interaction with other molecules is known from the prior art (Ku! Ebb IooShbe (1982), 1. Μοί. Vu1., 157: 105131, the work is mentioned in the present description by reference ) It is known that individual amino acids can be replaced by other amino acids with a similar hydropathic index while maintaining the biological activity of the protein. When making substitutions taking into account the hydropathic index, substitutions of amino acids are preferred whose hydropathic indices are in the range of ± 2, particularly preferred in the range of ± 1 and even more preferred in the range of ± 0.5.

It is also known from the prior art that substitutions of such amino acids can be effectively carried out on the basis of hydrophilicity, especially when the resulting biologically functional equivalent protein or peptide, as in the present case, is intended for immunological use.

As indicated in US Pat. No. 4,554,101, incorporated herein by reference, the highest average local hydrophilicity of a protein, determined by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with the biological properties of the protein.

As indicated in US Pat. No. 4,554,101, the following hydrophilicity values are characteristic of amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+ 3.0 ± 1); glutamate (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glut min (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

When making substitutions based on similar hydrophilicity values, amino acid substitutions are preferred whose hydrophilicity values are within ± 2, particularly preferred within ± 1 and even more preferred within ± 0.5.

US Pat. No. 4,554,101 also describes the identification and preparation of epitopes from primary amino acid sequences based on hydrophilicity. Using the methods set forth in US Pat. No. 4,554,101, a qualified person is able to identify epitopes in such amino acid sequences as the 8ΤΝΡΚ.8 sequences described herein. These regions are also referred to as nuclear epitope regions.

Numerous scientific publications are devoted to the prediction of the secondary structure and the identification of epitopes based on the analysis of amino acid sequences (Failures apb Gaktap (1974), Vyusbet18! Tu, 13: 2 222-245; Failures apb Gaktap (1978), Α6ν. Ephuto1. Ke1a !. Ageak Mo1 . Vu1., 47: 45-148; Failures of apb Gaktap, App. Ket. Vyusbet., 47: 251-276 and Failures of apb Gaktap (1979) Vyurbuk. 1., 26: 367-384, the works are mentioned in the present description in as links).

In addition, computer programs are currently available that help predict the antigenic regions and nuclear epitope regions of proteins. Examples are programs based on Jameson-Wolfe analysis (1 atecop apb \ Uo1G (1998), Sotry !. Arp1. Vyuksts 4 (1): 181-186 and \ Uo1G e! A1., (1988), Erase !. Arp1 Vyuks, 4 (1): 187-191, works are referred to in the present description by reference), the program РРР1о! ® (Вг! 1ад е! А1., (1990), САВ8, 6: 237-245 and АетЬещег е! A1., (1985), 8c1epse, 228: 740-742, works are mentioned in the present description by reference), and other new programs for predicting the quaternary structure of proteins (Reotote apb Vguap! (1993), Vyu esbpo1odu, 11 : 479-483, the work is mentioned in the present description by reference).

Conservative modifications of the amino acid sequence (and corresponding modifications of the nucleotide sequence encoding it) K1- [Su8 ^19- Suk ¹⁰³ ] -K2 and S.4 [Suk ³² - Suk ¹¹⁵ ] -K5 can lead to the formation of proteins with similar functional and chemical characteristics.

Conversely, significant modifications to the functional and / or chemical properties of Κ ₁ [Su8 ¹⁹ -Su8 ¹⁰³ ] -K and K2- [Su8 ^32- Suk ¹¹⁵ ] -K3 can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) polypeptide backbone structures in the region of substitution, for example, maintaining a flat or spiral structure; (b) changing the charge or hydrophobicity of the protein at the substitution site; or (c) adding a side chain. Naturally occurring residues are divided into several groups based on the general properties of the side chain:

1. Hydrophobic: Me !, A1a, Wa1, Leu, 11e;

2. Neutral hydrophilic: Suk, 8eg, T11g;

3. Sour: Acre, S1ts;

4. Basic: Akp, C1p, Ηίκ, Luk, Agd;

5. Aromatic: Tgr, Tug, Rye;

6. Residues affecting the orientation of the chain: C1u, Rgo.

Non-conservative substitutions may include replacing one of the group members with another. Such replacement residues can be introduced in the region of K ₁ - [Suk ^19- Suk ¹⁰³ ] -K2 and K4- [Suk ³² Suk ¹¹⁵ ] -K.5 homologous or non-homologous with other representatives of the ISE / tar receptor family.

Specific mutations in the sequences K1- [Suk ^19- Suk ¹⁰³ ] -K.2 and K_4- [Suk ^32- Suk ¹¹⁵ ] K ₅ include substitutions of a non-natural amino acid at the Ν-terminus, C-terminus, or anywhere in a protein that is modified the addition of Ν-linked or O-linked carbohydrate. Such modifications may be particularly useful since the addition of an amino acid (e.g., cysteine) can be advantageous in binding to a water-soluble polymer to form a derivative, as described below. See, for example, FIG. 5, where the natural AkP ¹⁰⁵ in kT№K-1 is replaced by Suk to facilitate the attachment of the polyethylene glycol molecule (example I).

In addition, the sequences K ₁ - [Suk ¹⁹ Suk ¹⁰³ ] -K2 and K4- [Suk ^32- Suk ¹¹⁵ ] -K5 can be modified by adding or removing the Ν-linked or O-linked glycosylation site. An asparagine-linked glycosylation site is a tripeptide sequence that is specifically recognized by the corresponding cellular glycosylation enzymes. These tripeptide sequences have the structure Akp-HaaT11G or Akp-Haa-8eg, where Xaa can be any amino acid except Prgo. Predicted or actual asparagine residues in kT№K-1 are in positions 14, 105 and 111. Predicted or actual asparagine residues in kT№K-1 are in positions 149 and 171.

A variety of amino acid substitutions or deletions can be made to modify or remove the Ν-linked or Bound glycosylation site, which will lead to a change in protein glycosylation.

In a specific embodiment, the variants are substantially homologous to the amino acid sequence K ₁ [Suk ^19- Suk ¹⁰³ ] -K2 or K4- [Suk ^32- Suk ¹¹⁵ ] -K5. The term “substantially homologous” means a degree of homology that generally exceeds 70%, more preferably exceeds 80%, even more preferably exceeds 90%, and most preferably is 95%. The homology percentage is calculated as the percentage of amino acid residues found in the smaller of the two sequences when compared with identical amino acid residues in the compared sequence, while four spaces per 100 amino acids can be entered to facilitate such a comparison as described by EauwoG w Abac oG Prgoesh 8es . | Iepse apb 81gis1ige, 5: 124 (1972), No. 1bop1 Vyusyetua1 Kekeagsb Eoipbabop, \ Wak11shd1op, E.S .. (the work is mentioned in the present description by reference). Truncated kT # KK variants that can be isolated by cross-reactivity with antibodies to the amino acid sequence 8EO GO N0: 2 and 8EO GO N0: 35, respectively, or those variants whose genes can be isolated by hybridization with DNA 8E0 GO N0, are also considered to be substantially homologous. : 1 or 8Е0 GO N0: 34 or their segments.

The following molecules are examples of kT№K.k according to the present invention:

MN2-MP8USRrSKU1NRr1 \ M81C- [Suk ¹⁹ Suk ¹⁰³ ] -RS-COOH (also designated as kT№K-1 2.6E / S105);

XH2-ME 8USROSK ¥ 1NPO \\ 81C- | C \ kSuk ¹⁰³ ] -E ^ 8b-COOH, (also denoted as kT№K-1 2.6E / C106);

\ H2- \ GO8 \ GROSCOW11 ^ 0 ^ 816-6 ^ Suk ¹⁰³ ] -E # COOH, (also denoted as kT№K-1 2.6E / 1005);

МН2-МУ1НРр1 \ М81С- [Suk ^19- Suk ¹⁰³ ] ЕЖ.'8Е-СОО (also designated as kT№K-1 2.3Ό / 68);

1MH2-M- [Suk ^19- Suk ¹⁰³ ] -E ^ 8T-COOH (also denoted as kT№K-1 2.3 6/618) and

1MN2-M818- [Suk ^19- Suk ¹⁰³ ] -E ^ 8T-COOH (also denoted as kT№K.-1 2.3Ό / 615) in both methionylated and non-methionylated forms, as well as their variants and derivatives.

Obtaining options truncated kT№Kk described in more detail below. Similar variants can be obtained by introducing appropriate nucleotide substitutions into DNA encoding truncated kT # K.k, or by chemical synthesis of 1n νίΙΐΌ desired truncated kT # K.k. As it is obvious to a qualified specialist, numerous combinations of deletions, insertions and substitutions can be made despite the fact that the final versions of truncated kT # Kk will retain biological activity.

Mutagenesis methods for replacing, inserting, or deleting one or more desired amino acid residues are well known in the art (for example, US Pat. No. 4,518,584, incorporated herein by reference). There are two main variables in the construction of each variant: the localization of the site of the mutation and the nature of the mutation. When constructing each variant, the localization of each mutation site and the nature of each mutation are determined by the biochemical characteristics that they want to change. Each mutation site can be modified individually or serially, for example, (1) by the initial replacement of any conservative residues and then, depending on the result, the introduction of a more radical replacement; (2) a deletion of a particular amino acid residue; or (3) an insertion of one or more amino acid residues near a selected site.

Based on the above description, a qualified specialist can obtain chemically modified derivatives of truncated 5ΤΝΕΚ.5. Using glycosylated, non-glycosylated or deglycosylated truncated 5ΤΝΕΚ.5 conjugates can be obtained. Usually use non-glycosylated truncated 5ΤΝΕΚ.5. Acceptable for derivatization of truncated 5ΤΝΕΚ.5 chemicals include water-soluble polymers.

Water-soluble polymers are preferred since the protein bound to them will not precipitate in an aqueous medium, in particular in a physiological medium of an organism. Preferably, the polymer should be pharmaceutically acceptable for the preparation of a therapeutic product or composition.

A qualified specialist will be able to choose the desired polymer taking into account the above considerations and depending on whether the polymer / protein conjugate is used therapeutically and, if so, taking into account the desired dose, circulation time and resistance to proteolysis. Convenient, clinically acceptable, water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyethylene glycol propionaldehyde, ethylene glycol / propylene glycol copolymers, monomethoxypolyethylene glycol, carboxymethyl cellulose, dextran, polyvinyl polyol di-polyol di-1,3-polyanoyl-1,3-polyanoyl-polyol di-polyanoyl-1,3-polyamide. 6-trioxane, a copolymer of ethylene and maleic anhydride, poly-β-amino acids (in the form of homopolymers or random copolymers), poly (p-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers olya (PPO) and other polyalkylene oxides, copolymers of polypropylene oxide / ethylene oxide, polyoxyethylene polyols (POC) (e.g. glycerol) and other polyoxyethylene polyols, polyoxyethylene sorbitol or polyoxyethylene glucose, colonic acids or other carbohydrate polymers, ficoll or dextran and mixtures of these compounds .

The concept of polyethylene glycol includes any form of polyethylene glycol used to derivatize other proteins, for example, mono (C ₁ -C ₁₀ ) alkoxy or aryloxy polyethylene glycol. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water.

Water-soluble polymers can have any molecular weight and be branched or unbranched. Typically, water-soluble polymers have an average molecular weight of about 2-100 kD (the term about means that in the preparation of the water-soluble polymer, some molecules will weigh more, some less, in comparison with the indicated molecular weight). The average molecular weight of each water-soluble polymer is in the range of about 5 to 50 kD, more preferably 12 to 40 kD, and most preferably 20 to 35 kD.

Typically, the higher the molecular weight and the more branched the polymer, the higher the polymer: protein ratio. Depending on the desired therapeutic profile (e.g., duration of controlled release; possible effects on biological activity; ease of handling; degree (or lack thereof) of antigenicity and other known effects of the water-soluble polymer on the therapeutic protein), polymers with different molecular weights may be used.

Water-soluble polymers must be attached to the protein, taking into account the possible effects on the functional and antigenic domains of the protein. In general, chemical derivatization can be carried out under any suitable conditions for the reaction of a protein with an activated polymer molecule.

Activating groups that can be used to convert the polymer into an active molecule include the following groups: sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5pyridyl.

Water-soluble polymers are usually attached to the protein via the α- or ε-amino groups of amino acids or a reactive thiol group, but it is also believed that the water-soluble group can be attached to any reactive group of the protein that is sufficiently reactive to join the water-soluble group under acceptable reaction conditions. Thus, a water-soluble polymer can be covalently bound to a protein via a reactive group, for example, a free amino or carboxyl group. Amino acid residues having a free amino group include lysine residues and Ν-terminal amino acid residues. Residues of aspartic acid, glutamic acid and C-terminal amino acid residues have a free carboxyl group. Residues with a free thiol group include cysteine residues.

Methods for the preparation of proteins conjugated to water-soluble polymers typically involve two steps: (a) reacting the protein with a water-soluble polymer under conditions under which the protein is coupled to one or more water-soluble polymers, and (b) isolating the reaction product. The reaction conditions for each case of conjugation can be selected among those known from the prior art and accordingly modified, but must necessarily exclude or limit such values of the reaction parameters (temperature, presence of solvents, pH values) that could inactivate the protein to be modified. In general, the optimal reaction conditions are determined for each specific case, based on known parameters and the desired result. For example, the higher the ratio of water-soluble polymer: protein conjugate, the higher the yield of the conjugated product. The optimal ratio (in terms of reaction efficiency, when there is no excess of unreacted protein or polymer) is determined by such factors as the desired degree of derivatization (for example, mono-, di-, tri-, etc.), molecular weight of the selected polymer, branching or unbranched polymer, as well as reaction conditions. The ratio of a water-soluble polymer (e.g., PEG) to protein typically ranges from 1: 1 to 100: 1. One or more purified conjugates can be obtained from each mixture by standard purification methods, which include, among others, dialysis, salting out, ultrafiltration, ion exchange chromatography, gel filtration and electrophoresis.

If desired, a protein chemically modified at the Ν-terminus can be obtained. A water-soluble polymer of the corresponding molecular weight, branching, etc., is selected, the ratio of the water-soluble polymer to the protein (or peptide) in the reaction mixture is determined, the type of reaction being carried out, and the method for isolating the selected protein chemically modified at the концу-terminus. A method for producing a drug chemically modified at the концу-terminus of a protein (for example, separating, if necessary, this form and other mono-derivatized forms) may be the purification of a Ν-terminal chemically modified protein from a population of chemically modified protein molecules. Selective термина-terminal chemical modification can be carried out by reductive alkylation, which uses the different reactivity of different types of primary amino groups (lysine versus Ν-terminal) available for derivatization in a particular protein. Under appropriate reaction conditions, significant selective derivatization of the protein at the Ν-terminus with a polymer containing a carbonyl group is achievable. For example, you can selectively attach a water-soluble polymer to the Ν end of the protein by carrying out the reaction at a pH value that allows you to take advantage of the difference between the pKa of the ε amino group of the lysine residues and the α-amino group of the термина-terminal protein residue. With such selective derivatization, the attachment of a water-soluble polymer to a protein is controlled: conjugation with the polymer occurs predominantly at the концу-end of the protein without significant modification of other reactive groups, such as amino groups of lysine side chains. For reductive alkylation, the water-soluble polymer may be one of the types described above and must have one reactive aldehyde to bind to the protein. Polyethylene glycol propionaldehyde containing a single reactive aldehyde may be used.

The present invention specifically claims a chemically derivatized protein, including its mono- or poly- (e.g., 2-4) PEG derivatives. Paging can be carried out using any reaction known in the art. Methods for producing a pegylated protein product typically involve two steps: (a) reacting the protein product with polyethylene glycol (e.g., a reactive ether or aldehyde derivative of PEG) under conditions in which the protein is coupled to one or more PEG groups, and (b) isolating the product ( or products) reactions. In general, the optimal reaction conditions are determined for each specific case, based on known parameters and the desired result.

There are a number of additional methods available to a qualified person (see, for example, application EP 0401384, referred to in the present description by reference; as well as Mike e! A1., (1992), Exp.Net! O1., 20: 10281035; PrgapsB (1992), Yosik op Sgo \\! 11 Ras! Og§, 3 (2): 4-10, (Riyejeb Lü Mebyspr !, Moip! Al Soig !, Rpeg your! Lape, Bopbop Ν20 OBO, IR); EP 0154316; EP 0 401 384; AO 92/16221; AO 95/34326 and other publications referenced herein).

In particular, pegylation can be carried out by acylation or alkylation reactions with a reactive polyethylene glycol molecule. Thus, the pegylated proteins in which PEG groups are attached via acyl or alkyl groups are also contemplated as protein products of the invention. These products may be mono-pegylated or poly-pegylated (for example, contain 2-6, and preferably 2-5, PEG groups). PEG groups are usually attached to the protein via the α or ε-amino groups of amino acids, but it is also believed that PEG groups can be attached to any amino group of the protein that is sufficiently reactive to attach the PEG group under acceptable reaction conditions.

When pegylation by acylation, the reaction of active ether derivatives of polyethylene glycol (PEG) with a protein usually occurs. For the acylation reaction, the selected polymer (or polymers) must have one reactive ester group. For the pegylation reaction, any of the known or subsequently found reactive PEG molecules can be used. A preferred activated PEG ester is PEG esterified with рок hydroxysuccinimide (ΝΗ 8). It is believed that the term acylation includes, without limitation, the following types of bonds between a therapeutic protein and a water-soluble polymer such as PEG: amide, carbamate, urethane, and the like (see C11ato (1994), Soysid! E Syssh .. 5 (2) : 133-140). The reaction conditions can be selected from the pegging reaction conditions known from the prior art and modified accordingly, but they must exclude such reaction parameters (temperature, presence of solvents, pH values) that could inactivate the protein to be modified.

Pegylation with acylation typically results in a pilipaged protein. Preferably, the binder is an amide. It is also preferred that the resulting product is substantially (e.g.> 95%) mono-, di-, or tripered. However, forms with a higher degree of pegylation can form, the number of which depends on the specific reaction conditions. If desired, more purified pegylated forms can be separated from the mixture (in particular, unreacted PEG) by standard purification methods, which include, among others, dialysis, salting out, ultrafiltration, ion exchange chromatography, gel filtration and electrophoresis.

Pegylation by alkylation usually requires the reaction of a terminal aldehyde derivative of PEG with a protein in the presence of a reducing agent. For the reductive alkylation reaction, the selected polymer (s) must have one reactive aldehyde group. An example of a reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or its mono-C ₁ -C ₁₀ alkoxy or aryloxy derivatives (see US Pat. No. 5,252,714, incorporated herein by reference).

Pegylation by alkylation can also result in a poly-pegylated protein. It is possible to select reaction conditions for the preferred pegylation of only α-amino groups at the Ν-terminus of the protein (i.e., the production of a monopaginated protein). In monopegging and polypegging, PEG groups preferably bind to the protein via —CH ₂ —YN groups. Given the role of the —CH ₂ group, this type of bond is referred to as an alkyl bond.

Reductive alkylation to obtain a substantially homogeneous population of a monopolymer / protein product typically involves two steps: (a) reacting the protein with a reactive PEG molecule under reductive alkylation at a pH value that selectively modifies the α-amino groups at the amino terminus of the protein, and (b) isolating product (or products) of the reaction. Derivatization by reductive alkylation takes advantage of the difference between the pKa of the amino groups of the lysine residues and the α-amino group of the термина-terminal protein residue (pKa is a pH value at which 50% of the amino groups are protonated and 50% are not).

The reaction is carried out at a pH value that allows you to take advantage of the difference between the pKa of the ε-amino group of the lysine residues and the α-amino group of the Ν-terminal protein residue. In general, if the pH value is lower, a larger excess of polymer is required compared with protein (i.e., the less available “terminal reactive α-amino groups, the more polymer is required to achieve optimal conditions). If the pH value is higher, then the ratio of polymer to protein does not have to be so high (i.e. more reactive groups are available, therefore less polymer molecules are needed). For the purposes of the present invention, the pH is usually in the range of 3-9, preferably 3-6. For reductive alkylation, the reducing agent must be stable in aqueous solution, and it is preferable to reduce only the Schiff base formed in the initial stages of reductive alkylation. Suitable reducing agents may be selected from the group consisting of sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and pyridine borane. A particularly convenient reducing agent is cyanoborohydride. Other reaction parameters, such as solvent, reaction time, temperature, and product purification method, are determined specifically for each case based on published information on protein derivatization with water-soluble polymers.

With such selective derivatization, the attachment of a water-soluble polymer (containing such a reactive group as an aldehyde) to the protein is controlled: conjugation with the polymer occurs predominantly at the концу-end of the protein without significant modification of other reactive groups, such as amino groups of the lysine side chains. A formulation typically consists of more than 90% monopolymer / protein conjugate, more typically more than 95% monopolymer / protein conjugate, with the remainder of the molecules not reacting (i.e., a protein without polymer groups).

In a specific embodiment, unbranched monomethoxypolyethylene glycolaldehyde with an average molecular weight of about 20 kD or about 33 kD (i.e. between 30 and 35 kD), or a quaternary butylpolyethylene glycolaldehyde with an average molecular weight of about 33 kD (i.e. between 30 and 35 kD ) conjugated by reductive alkylation with δΤΝΡΡ-Ι 2.6Ό / Ν105.

Pegging can also be carried out using water-soluble polymers having at least one reactive hydroxy group (e.g., polyethylene glycol). In this case, the water-soluble polymer reacts with a reagent having a carbonyl, nitrile or sulfone group to convert the hydroxyl group to a Michael reactive acceptor, thereby forming an activated linker suitable for modifying various proteins to obtain improved biologically active conjugates. Reactive carbonyl, nitrile or sulfone means a carbonyl, nitrile or sulfonic group to which a second carboxylic acid group is attached to form a reactive site for thiol-specific binding of the second carbon of a carbonyl, nitrile or sulfonic group (AO 92/16221).

Activated linkers can be monofunctional, bifunctional or multifunctional. Reagents having reactive sulfonic groups that can be used to carry out these methods include, but are not limited to, chlorosulfone, vinyl sulfone, and divinyl sulfone.

In a specific embodiment, the water soluble polymer is activated by a Michael acceptor. Application AO 95/13312 describes, for example, aya, water-soluble sulfone-activated polyethylene glycols that bind highly selectively to thiol groups instead of amino groups on molecules or surfaces. These derivatives

PEGs are resistant to hydrolysis when they are in the aqueous medium for a long time and to pH 11 or lower, and also form bonds with molecules, which leads to the formation of hydrolytically stable conjugates. The combination of PEG and a biologically active molecule is achieved by linking the sulfone group and the thiol group, which leads to the formation of the structure PEC-8O ₂ -CH ₂ -CH ₂ -8-A, where A denotes a biologically active molecule, and where the sulfonic group is represented by vinyl sulfone or active ethyl sulfone. Especially useful are two homobifunctional derivatives, namely PEG-bis-chlorosulfone and PEG-bisvinylsulfone.

Published international application No. 96/19459, incorporated herein by reference, describes methods for preparing sulfon-activated linkers by preparing a compound having a reactive hydroxyl group and converting the hydroxyl group to a Michael reactive acceptor to form an activated linker. At the same time, tetrahydrofuran (THE) is used as a solvent for the conversion. The published international application υδ 96/19459, incorporated herein by reference, describes a process for purifying activated linkers using hydrophobic interaction chromatography to separate linkers based on size and function of the end group.

In particular, the present invention contemplates the following molecules expressed in prokaryotes that are chemically derivatized with the addition of mono or poly (i.e. 2-4) PEG groups: δΤΝΡΡ-Ι 2.6B / C105, δΤΝΡΚ-Ι 2.6B / C106, δΤΝΡΚ -Ι 2.6Ό / Ν105, δΤΝΡΚ-Ι 2.3Ό / 68, δΤΝΡΚ-Ι 2.3Ό / 618 and 8ΤΝΕΚ.-Ι 2.3Ό / 615, in methionylated or non-methionylated form, as well as their variants and derivatives.

Polyvalent forms.

Polyvalent forms can be constructed, i.e. molecules containing more than one active principle. In one embodiment, a molecule may have multiple tumor necrosis factor binding sites for ligand ΤΝΡ (i.e., a combination of truncated products δΤΝΕΚ.). Additionally, the molecule may possess at least one tumor necrosis factor binding site and, depending on the desired characteristics of the multivalent form, one binding site of another molecule (for example, a combination of at least one truncated product δΤΝΡΚ. And at least one interleukin-1 receptor antagonist -On), as described below).

In a specific embodiment, the polyvalent forms can be constructed, for example, by chemically combining at least one truncated product δΤΝΕΚ. with any clinically acceptable linker (i.e., a water-soluble polymer as described above). In principle, the linker should not introduce new immunogenicity, or, due to the presence of new amino acid residues, change the hydrophobicity or balance of charges in the structure, which could negatively affect its biodistribution and clearance.

Such polymers, when used as linkers, can be homopolymers, random or block copolymers and terpolymers based on the above monomers, with a straight or branched chain, with or without substitutions. The polymer can have any length and molecular weight, but these characteristics can affect biological properties. The average molecular weights of the polymers, which are especially useful for lowering the clearance rate in pharmacological applications, range from 2,000 to 35,000 daltons. In addition, to optimize or impart the desired biological activity, the length of the polymer may vary.

Activating groups that can be used to bind a water-soluble polymer to two or more proteins include sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5pyridyl.

In a specific embodiment, a bifunctional or multifunctional activated linker having at least one Michael reactive acceptor can be obtained in accordance with US patent application No. 08 / 473,809 and purified in accordance with US patent application No. 08 / 611,918.

Active principles may be coupled using conventional coupling methods (see published international applications AO 92/16221 and AO 95/34326, referred to in the present description by reference). In addition, application AO 92/16221 describes the preparation of various dimerized δ молекул-ΤΝΕΒ inhibitor molecules i.e. dimerized with 105 δΝΤΈΒ-Ι. An example is polyvalent proteins that bind tumor necrosis factor and having the formula (δΤΝΕΚ-Ι 2.6Ό / Ο106) ₂ (20 kLa REO), which are described in example I.

Alternatively, the bivalent molecule may consist of two tandem repeats of truncated δΤΝΕΒ products separated by a polypeptide linker.

The design of the polypeptide linkers is similar to the construction of the insertion of short-loop sequences between the domains of the constructed non-protein proteins (Miyet (1988), ΤΊΒ8, 13: 260-265 and Vedap apb OeOtabo (1988), 8c1epse, 241: 976-978, the works are mentioned in the present description in as links). It was shown that a linker acceptable for single chain antibodies is effective for the preparation of dimeric forms of recombinant human κΤΝΕΒ-ΙΙ (Newe e1 a1., (1996), Cy1okshe, 8 (5): 365-370, the work is mentioned in the present description by reference ) Several different linker constructs have been obtained that are useful for use with antibodies; the most functionally active linkers vary in size from 12 to 25 amino acids (amino acids with non-reactive side groups, such as alanine, serine and glycine), which together constitute a hydrophilic sequence and have few oppositely charged residues, which increases the solubility of the molecule and makes it flexible (APP Eiri (1991), Méliob§: A Sochrashop 1o Méliob and Ep7ushoodu, 2: 97-105 and Vydlbo e1 a1., (1993), Nshipok, 150: 469-479, the works are mentioned in the present description by reference).

In a specific embodiment, the truncated δΤΝΡΒδ can be chemically attached to biotin, and then the biotin / truncated δΤΝΤΒδ conjugates are coupled to avidin, resulting in the formation of tetravalent avidin / biotin / truncated δΤΝΕΚ, δ molecules. Truncated δΤΝΡΒδ can also be covalently linked to dinitrophenol (ΌΝΡ) or trinitrophenol (ΤΝΡ), and the resulting conjugates are precipitated with anti-ΌΝΡ or anti-ΤNΡ-IdM to form decameric conjugates with a valency of 10 with respect to ΤΝΕ-binding sites.

According to another embodiment of the invention, recombinant fusion proteins containing truncated κΤΝΕΒ can be obtained, while in each chimeric molecule the κ последовательность sequence, as described above, is replaced by variable domains of heavy or light chains and contains full or partial partial domains (but at least one constant domain) of heavy or light chains of human immunoglobulins.

For example, each such chimeric fusion protein (truncated δΤΝΤΒ / ^ Ο1) can be obtained from two chimeric genes: truncated δΤΝΕΒ / kappa 1d human light chain (truncated δΤΝΕΒ / Sk) and truncated KEPEV / gamma 1d human heavy chain (truncated MLEV / Sd -!). After transcription and translation of two chimeric genes, as described below, gene products can be assembled into a single chimeric molecule in which truncated κΤΝΕΒκ are bivalently represented. Additional details of the construction of such chimeric molecules are described in US patent 5,116,964, published international application AO 89/09622, published international application AO 91/16437 and EP 315062, referred to in the present description by reference.

According to one embodiment of the invention, recombinant fusion proteins with truncated δΤΝΕΒδ specificity can be obtained, with each recombinant chimeric molecule containing a δΤΝΕΚ .. sequence as described above, as well as at least a portion of the osteoprotegrin (OPC) region 186-401, as described in the application for European patent No. 96309363.8.

Polynucleotides.

The present invention also relates to polynucleotides encoding truncated 5ΤΝΕΚ.5. Based on the present description and using a universal codon table, any qualified specialist can easily determine all nucleotide sequences encoding the amino acid sequences of truncated 5ΤΝΕΚ.5. Preferred nucleotide sequences include those polynucleotides that encode δΤΝΕΒ-Ι 2.6E / C105, δΤΝΕΒ-Ι 2.6O / C106, δΤΝΕΚ-Ι 2.6Ό / Ν105, δΤΝΕΚ-Ι 2.3Ό / 68, δΤΝΕΚ-Ι 2.3Ό / 618 and δΤΝΕΚ-Ι 2.3Ό / 615. Examples of various polynucleotides are shown in FIG. 2, 3, 4, 5, 6 and 7.

Recombinant expression methods, as described below, can be used to prepare such polynucleotides and express the proteins encoded by them. For example, by introducing a nucleotide sequence encoding a truncated δΤΝΕΚ. in the appropriate vector, a qualified person can easily obtain large quantities of the desired nucleotide sequence. The sequences can then be used to prepare hybridization probes or amplification primers. Alternatively, a polynucleotide encoding a truncated δΤΝΕΚ. Can be introduced into the expression vector. When the expression vector is introduced into the corresponding host cells, large quantities of the desired truncated δΤΝΕΚ can be obtained.

As can be seen from the following description, there are numerous host / vector systems suitable for obtaining the desired nucleotide sequences and / or production of the desired proteins. These include, but are not limited to, plasmid, viral, and insertion vectors and prokaryotic and eukaryotic host cells. A qualified specialist can adapt the host / vector system for the growth and expression of heterologous DNA in order to obtain or expression of the sequences claimed in the present invention.

In addition, as follows from the present description and which is obvious to a qualified specialist, nucleotide sequences include degenerate nucleotide sequences encoding truncated 5ΤΝΕΚ.5 and having the structure shown in the drawings, as well as nucleotide sequences that hybridize (preferably under stringent hybridization conditions) with data nucleotide sequences [MashDK c1 a1., (1982), Mo1ci1a1 Cl1aaaaa (Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ...) to ... 387389]. An example of hybridization under stringent conditions is hybridization at 4 x 88 ° C at 6267 ° followed by washing at 0.1 x 88 ° C at 62-67 ° for one hour. Alternatively, stringent hybridization can be carried out in 45-55% formamide, 4 x 88C at 40-45 °. Also included within the scope of the present invention are DNA sequences that hybridize to the nucleotide sequences shown in FIG. 1 and 9, under mild hybridization conditions and which encode truncated 8ΤΝΕΚ.8.

Examples of such mild hybridization conditions are hybridization at 4 x 88 ° C at 45-55 ° C or hybridization at 30-40% formamide at 40-45 °.

Recombinant DNA constructs, including vector DNA along with DNA sequences encoding truncated 5ΤΝΕΚ5, are also claimed according to the present invention. In each of these DNA constructs, nucleotide sequences encoding truncated 5ΤΝΕΚ.5 (with or without signal peptides) are functionally linked to acceptable regulatory sequences capable of directing replication and or expression of truncated 5ΤΝΕΚ.5 in a specific host.

Recombinant expression.

Obtaining polynucleotides.

Nucleotide sequences encoding truncated δΤΝΡΡδ can be obtained in various ways, including (but not limited to) chemical synthesis, screening of genomic and cDNA libraries, screening of expression libraries and / or PCR amplification of cDNA. These methods, as well as others suitable for the isolation of nucleotide sequences, are known from the prior art and are described in the following manuals: 8AbGooc1a1C1, B1C1a1C1C1c2c: A6a0a0a0a1aaa1a1aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa; (1989); Ai8bie1 e1 a1., Fuck, siggep! Prigo1osok w Mo1esi1ag Vu1odu, Siege! Rgo1osok Rgekk, (1994); and Vegdeg apb K1tte1, MeSzobk and Ephutoodu: Sbbe 1o Mo1esi1ag S1oshpd Τesbn ^^ ie8, Uo1.152, Asabetk Rgekk, Sch., 8ap O1edo, SA, (1987), referred to in the present description by reference.

The chemical synthesis of nucleotide sequences encoding truncated 8-8 is carried out in accordance with methods well known in the art, described, for example, in Epdek e1 a1., (1989), Ref. C1. SHD. Eb., 28: 716-734 and ^ e11k e! A1., (1985), Sepe, 34: 315, referred to in the present description by reference. These methods include, among others, phosphotriether, phosphoramidate and H-phosphonate methods for the synthesis of nucleotide sequences. Large nucleotide sequences, for example, longer than 100 nucleotides, can be synthesized in several fragments. Then the fragments are subsidized with each other and a nucleotide sequence encoding truncated 5ΤΝΡΚ.5 is obtained. A preferred method is synthesis on a polymer substrate using standard phosphoramidate chemistry.

Alternatively, the desired nucleotide sequences can be obtained by screening the corresponding cDNA libraries (for example, libraries obtained from one or more tissues in which the protein is expressed) or genomic libraries (libraries obtained from total genomic DNA). The source for the cDNA library is usually any type of tissue in which the desired protein is expressed in acceptable amounts. Typically, the source of the genomic library is any tissue or tissues of a mammal or animal of another species that has a gene encoding a truncated δΤΝΡΚ.

The library can be screened for DNA encoding truncated δΤΝΡΚ using one or more nucleotide probes (oligonucleotides, cDNA or genomic DNA fragments that have a sufficient level of homology with cDNA or the cloned gene) that will selectively hybridize with cDNA or the gene (s) present (s) in the library. The samples used for such screening are usually a small fragment of a DNA sequence of the same or similar biological species as the animal species from which the library was obtained. Alternative probes may be degenerate, as discussed below.

Typically, a library is screened by annealing an oligonucleotide probe or cDNA with library clones under conditions of such stiffness that prevents non-specific binding, but makes it possible to bind to the probe or primer those clones that have a significant level of homology with the probe or primer. Typically, the stringency parameters of hybridization and subsequent washings depend on the size (i.e., the number of nucleotides) of the cDNA or oligonucleotide probe, as well as whether the probe is degenerate. When compiling a hybridization mixture, the likelihood of clone identification is also taken into account (i.e., a cDNA or genomic library is screened).

When a DNA fragment (such as cDNA) is used as a probe, the hybridization conditions are selected as described by Aikbie! e! A1., ebk. Kirga. After hybridization, the library containing blot is washed under conditions of suitable stiffness, which depends on factors such as the size of the probe, the expected homology of the probe and clone, the nature of the library being scanned, the number of clones to be scanned, etc. The following are examples of hard washing solutions, which usually have low ionic strength and are used at relatively high temperatures: 1) 0.015M No. C1, 0.005M No. 1-citrate and 0.1% 8Ό8 at 55-65 ° C; 2) 1 mM No. ₂ 40, 40 mM NaHΡO ₄ , pH 7.2, and 1% 8Ό8 at 40-50 ° C; 3) 0.2 x 88С, 1% 8Ό8 at 50-65 ° С.

Various exemplary washing protocols are known under stringent conditions when oligonucleotide probes are used to screen cDNA or genomic libraries. For example, one of the protocols uses 6 x 88С with 0.05% sodium pyrophosphate at a temperature of 35 ° C to 63 ° C, depending on the length of the probe. For example, a probe consisting of 14 bases is washed at 35-40 ° C, from 17 at 4550 ° C, from 20 at 52-57 ° C and from 23 at 57-63 ° C. With enhanced non-specific (background) binding, the temperature can be increased by 2-3 ° C. Another protocol uses tetramethylammonium chloride (TMAC) for laundering. One of the hard washing solutions contains 3 M TMAS, 50 mM TP8-HC1, pH 8.0, and 0.2% 8–8.

Another convenient method for obtaining the desired nucleotide sequence is polymerase chain reaction (PCR). In this case, cDNA is obtained on a poly (A) + RNA or total RNA template using the reverse transcriptase enzyme. Two primers (oligonucleotides) are added to the cDNA, usually complementary to two different regions of the cDNA encoding truncated δΤΝΡΚ .. as well as a polymerase, for example Tad polymerase. In this case, the polymerase amplifies the cDNA region between the primers.

Oligonucleotide sequences selected as probes or primers should be of adequate length and unambiguous enough to minimize the nonspecific binding that may occur during library screening or PCR amplification. The actual sequence of probes or primers is usually based on conserved or highly homologous sequences or sites. Additionally, probes or primers can be completely or partially degenerate, i.e., they are a mixture of probes / primers that collectively encode the same amino acid sequence, but use different codons for this. An alternative to the preparation of degenerate probes is the introduction of inosine into some or all codons, the position of which varies depending on the species. Oligo nucleotide probes or primers can be obtained by chemical synthesis of DNA, as described above.

As indicated above, variant sequences are those natural (e.g. allelic variations) or synthetic sequences that contain one or more nucleotide substitutions, deletions or insertions, as compared to the sequence shown in FIG. 2, 3, 4, 5, 6 and 7, which leads to the expression of amino acid sequence variants compared to the wild-type amino acid sequence. Obtaining synthetic mutant sequences is well known in the art and is described, for example, in the works of SATLOGO e! A1., (1989), Zirga and \ Ue11k e! A1., (1985), Sepe, 34: 315, referred to in the present description by reference.

Vectors.

DNA encoding truncated δΤΝΕΚδ is inserted into vectors for further cloning (DNA amplification) or expression. Commercially available vectors can be used or the vector is designed specifically. The choice and design of the appropriate vector will depend on the following factors: 1) whether the vector will be used for DNA amplification or for DNA expression; 2) on the size of the DNA to be inserted into the vector, and 3) on which cell hosts will be transformed by the vector.

In any vector, the nucleotide sequence encoding the desired protein is functionally linked to one or more regulatory sequences that are capable of directing, controlling, and generally affecting the expression of this protein in specific host cells. Each vector contains various components depending on its purpose (DNA amplification or DNA expression) and compatibility with certain host cells. A vector usually contains the following components (without limitation to those listed): signal sequence, start of replication, one or more selective (e) or marker (s) gene (s), promoter, enhancer, transcription termination sequence and the like. These components can be obtained from natural sources or synthesized by known methods.

Suitable prokaryotic cloning vectors include bacteriophages, in particular lambda phage derivatives, E. coli plasmids (for example, pBK322, co1 E1, pIS, E-factor and plasmid derivatives B1ie8spr ® ® (8! Ha! Adepe, ba1o11a, CA)). Other expression vectors can be used, numerous types of which are known in the art and which are compatible with the host cells described below.

Signal sequence.

A nucleic acid encoding a signal sequence can be inserted into the 5'-terminal portion of a sequence encoding a truncated δΤΝΕΚ., I.e. it may be a component of the vector, or it may be part of a nucleic acid encoding a truncated δΤΝΕΚ. embedded in the vector. Nucleic acids encoding the native signal sequences δΤΝΕΚ-Ι and δΤΝΕΚ-ΙΙ are known in the art (EP 393438 and EP 422339).

Start of replication.

Cloning and expression vectors usually include nucleotide sequences that enable the vector to replicate in one or more types of host cells. In the case of a cloning vector, such a sequence allows the vector to replicate independently of the host chromosomal DNA and includes the start of replication or autonomously replicating sequences. Such sequences are well known. The onset of replication of plasmid pBK322 is acceptable for most gram-negative bacteria, and various other replication origins (e.g., 8U40 virus, polyoma viruses, adenovirus, U8U, or ASU) are suitable for vectors intended for cloning in mammalian cells. Typically, the onset of replication is not necessary for vectors designed for expression in mammalian cells (for example, the onset of 8U40 replication is often used only because it contains an early promoter).

Selective gene.

Cloning and expression vectors usually contain a selective gene. This gene encodes a marker protein necessary for the survival or growth of a transformed host cell when grown on selective culture medium. Cells that are not transformed by the vector will not contain a selective gene and therefore will not survive in this culture medium. Typical selective genes encode proteins that a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline; b) complement auxotrophic deficiencies or c) provide critical nutrients inaccessible from the culture medium.

Other selective genes are used to amplify expressed genes. Amplification is a process in which the genes encoding the desired protein and the genes necessary for cell growth are repeated in tandem again and again on the chromosomes of successive generations of recombinant cells. Examples of selective markers suitable for mammalian cells include dihydrofolate reductase (ΌΗΕΚ) and thymidine kinase. Transformed mammalian cells are grown under selective pressure at which, thanks to the marker transferred by the vector, only true transformants survive. Selective pressure is provided by culturing the transformed cells under conditions of a successively increasing concentration of the selective agent in the medium, which leads to amplification of the selective gene and DNA encoding truncated 5ΤΝΡΚ.5. As a result, increased amounts of truncated 5ΤΝΡΚ.5 are synthesized on amplified DNA.

For example, cells transformed with the selective ΌΗΡΚ gene were first isolated by culturing the entire set of transformants in a culture medium with methotrexate, a competitive antagonist of ΌΗΡΚ. Suitable host cells for the wild type are Chinese hamster ovary cells, which are deficient in their activity (see, for example, Iglai and Apb Ott (1980), Proc. Ba! 1. Lsb. Δοΐ. I. 8.L., 77 ( 7): 4216-4220, referred to in the present description by reference). Transformed cells are then cultured at increasing concentrations of methotrexate. This leads to the synthesis of multiple copies of the ΌΗΡΚ gene and, concomitantly, multiple copies of other DNA present in the expression vector, for example, DNA encoding a truncated δΤΝΡΚ.

Promoter.

Both expression vectors and cloning vectors usually contain a promoter that is recognized by the host organism and is functionally linked to a nucleotide sequence encoding a truncated δΤΝΡΚ Promoter is an untranslated sequence located above the (5 ') - start codon of the structural gene (usually in the range of 100-1000 base pair), which controls the transcription and translation of a specific nucleic acid sequence, in particular, encoding a truncated δΤΝΡΚ. Promoters are usually divided into two classes: inducible promoters and constitutive promoters. Inducible promoters induce increased transcription of DNA under their control in response to any changes in cultivation conditions, for example, temperature shift or the presence or absence of any nutrient. A large number of promoters are recognized by a variety of host cells. The promoter can be functionally linked to DNA encoding truncated δΤΝΡΚ by restriction removal of the existing promoter from the DNA and insertion of the desired promoter sequence into the vector. The native promoter sequence δΙ-Ι or the promoter sequence δΤΝΡΚ-ΙΙ can be used to control the amplification and / or expression of DNA encoding truncated δΤΝΡΚ. A heterologous promoter, however, is more preferable because it allows a higher level of transcription and provides a higher yield of expressed protein than the native promoter, while it is compatible with the selected cellular expression system. For example, any of the native promoter sequences of other members of the ΝΟΡ / семейства family can be used to control amplification and / or expression of DNA encoding truncated 8ΤΝΡΚ.

Promoters suitable for use in prokaryotic hosts include the beta-lactamase and lactose promoter; alkaline phosphatase promoter and tryptophan (! rg) promoter system, bacterial luminescence gene (ΗχΚ) system, and hybrid promoters such as! as promoter. Other known bacterial promoters are also acceptable. Their nucleotide sequences are published, which allows a qualified specialist to ligate them with the desired DNA sequences using linkers or adapters necessary to create the desired restriction sites.

Convenient promoter sequences for use in yeast cells are also known in the art. Promoters are known for use in mammalian host cells, including promoters isolated from virus genomes such as polyoma virus, smallpox virus, adenovirus (e.g., type 2 adenovirus), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis virus B and most preferably monkey virus 40 (8Y40). Other convenient mammalian promoters include heterologous mammalian promoters, for example, heat shock promoters and the actin promoter.

Enhancer element.

An enhancer sequence may be introduced into the vector to enhance transcription of higher DNA sequences encoding truncated δΤΝΡΚ in higher eukaryotic cells. Enhancers are cis-acting DNA elements, usually 10-300 base pairs long, that affect the promoter, enhancing transcription. Enhancers are relatively independent of orientation and position. They can be located both from the 5'-end and from the 3'end of the transcription unit. Yeast enhancers are preferably used in conjunction with yeast promoters. The enhancer sequences of a number of mammalian genes are known (e.g., globin, elastase, albumin, alpha-fetoprotein and insulin). Usually, however, viral enhancers are used. Examples of enhancer elements activating eukaryotic promoters are the 8ν40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma virus enhancer, and the adenovirus enhancer. While the enhancer can be inserted into the vector both at the 5'-position and the 3'-position with respect to the DNA fragment encoding the truncated κΊΝΕΚ, it usually occupies the 5'-position relative to the promoter.

Transcription termination.

Expression vectors used in eukaryotic host cells typically contain the sequences necessary for termination of transcription and stabilization of mRNA. Similar sequences are usually found in 5'- (and sometimes 3'-) untranslated regions of eukaryotic DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding truncated κΊΝΕΚ.

Vector design.

The construction of acceptable vectors containing one or more of the above components (together with the semantic regions encoding the truncated kT # K) can be carried out by standard ligation methods. Isolated plasmids or DNA fragments are cut, cut and re-ligated in the correct order to obtain the desired vector. In order to ensure that the correct sequence is constructed, the E. coli is transformed with the ligation mixture and the transformants are selected by known methods, as described above. Then a vector is isolated from transformants, subjected to restriction analysis and / or sequencing to confirm the presence of the desired construct.

A vector that provides transient expression of DNA encoding truncated kT # K in mammalian cells can also be used. Generally speaking, transient expression involves the use of an expression vector that is capable of efficiently replicating in the host cells, so that many copies of the expression vector are accumulated in the cells and then large amounts of the desired protein encoded by the expression vector are synthesized. Each transient expression system, consisting of an appropriate expression vector and host cells, provides convenient identification of proteins encoded by cloned DNA, as well as the ability to quickly screen these proteins for desired biological or physiological properties, for example, allows identification of a biologically active variant of truncated cTK .

Host cells.

Any recombinant host cells containing the nucleotide sequence necessary for expression of the desired protein are also included in the scope of the present invention. Examples of prokaryotic and eukaryotic host cells are cells of bacteria, mammals, fungi, insects, yeast or plants.

Prokaryotic host cells include (but are not limited to) eubacteria, such as Gram-negative or Gram-positive organisms (e.g. E.co11 (HB101, OH5a, 10 and MC1061); VasB1, such as B. kb! Shk; Rkeibotopak, such as R.aegidtoka; 8! Ger! Flavored crr .; 8a1topope11a! Urbutigsht or 8egga! 1a tagseksapk. An example of a specific embodiment of the invention is the expression of the desired protein in E.co11.

In addition to prokaryotic host cells, eukaryotic microorganisms such as filamentous fungi and yeast can serve as suitable hosts for expression of the desired protein. Sassagotus cecum \ zk1ae, or ordinary baker's yeast, are the most widely used lower eukaryotic hosts, however, representatives of other genera, species and strains are well known and widely available.

Truncated kT№K can be expressed in glycosylated form in any host cells derived from a multicellular organism. Such host cells provide complex protein processing and glycosylation. In principle, you can use the culture of any eukaryotic cells of vertebrates and invertebrates, including plant and insect cells. An example of a specific embodiment of the invention is the expression of the desired protein using a baculovirus system in insect cells.

The growth of vertebrate cells in culture (tissue culture) is a well-known approach and, therefore, vertebrate cells can be used as host cells. Examples of useful mammalian cell lines include, but are not limited to, Ον1 monkey kidney cells transformed with 8ν40 (CO8-7), human embryo kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture), hamster kidney cells and Chinese hamster ovary cells. Other acceptable mammalian cell lines include, but are not limited to, Heaven cells. murine L-929 cells, 3T3 cell lines obtained from mice of 8 ^ 1cc, Ba1b-c or а, as well as hamster cells of BHK or NaK. An example of a specific embodiment of the invention is the expression of the desired protein in CO8 cells.

Host cells can be transfected and preferably transformed with the desired nucleic acid under appropriate conditions for expression of the nucleotide sequence. The methods for selecting the necessary host cells, as well as the methods of transformation, cultivation, amplification, screening, as well as the methods for obtaining and purifying the product, are well known in the art (Seyyip aib 8ashgook (1981), No. 1ite, 292: 620-625, or, alternatively, KaiGshap e1 a1., (1985), Mo1. Ce11. Bu1. 5 (7): 1750-1759, or US patent No. 4,419,446; these sources of information are given here by reference). For example, for mammalian cells without a cell wall, calcium phosphate precipitation can be used. Electroporation, microinjection and other known methods may be used.

In addition, it is possible to obtain the desired protein by homologous recombination or by recombinant nucleic acid methods using control elements introduced into cells already containing DNA encoding truncated δΤΝΕΚ. Homologous recombination is a method originally designed to target (target) genes for induction or correction mutations in transcriptionally active genes (Kisebarab (1989), Year Ιη Νιιοί. As1b Kek. Aib Mo1. ΒίοΙ., 36: 301; the work is referred to in the present description as a reference lki). The initial approach was developed as a method for introducing specific mutations into specific areas of the mammalian genome (Shotaya e1 a1., (1986), Ce11, 44: 419-428; Lupt aib SaressA (1987), Ce11, 51: 503-512 and BoeOsytap e1 a1 ., (1988), Proc. No. 11. Asab. Δά.υ.δ.Α., 85: 85838587; all works are mentioned in the present description by reference) or for the correction of specific mutations in defective genes (BoeEsaytai e1 a1., (1987), No. 11ige, 330: 576-578; the work is also referred to in the present description by reference). Examples are the methods described in US Pat. No. 5,272,071; AO 92/01069 applications; AO 93/03183; AO 94/12650 and AO 94/31560, referred to in the present description by reference.

Through homologous recombination, the DNA sequence that must be inserted into the genome can be directed to a specific region of the gene of interest. This is achieved by attaching it to the targeting DNA. Targeting DNA is DNA that is complementary (homologous) to a certain region of genomic DNA. Small fragments of targeting DNA that are complementary to a specific region of the genome come into contact with the parent DNA strand during the DNA replication process. A common property of DNA transferred into the cell is the ability to hybridize and therefore to recombine with other fragments of endogenous DNA through areas of general homology. If such a complementary strand is attached to an oligonucleotide containing a mutation or other DNA sequence, it also integrates into the newly synthesized strand as a result of recombination. As a result of the reading check function, it is likely that the new DNA sequence will serve as a template. Thus, the transferred DNA is integrated into the genome.

If the sequence of a particular gene is known, in particular the nucleotide sequence encoding the desired protein, it is possible to synthesize (or obtain in another way, for example, by appropriate restriction of native DNA at specific sites that limit the desired region) the expression control sequence (DNA fragment complementary to the selected gene region) . Such a fragment serves as a targeting sequence when introduced into the cell and will hybridize with the homologous region of the genome. If such hybridization occurs during DNA replication, then such a DNA fragment and any additional sequences attached to it will work as Okazaki fragments and will be embedded in the newly synthesized daughter DNA strand.

Attached to the targeting DNA fragments are those regions of DNA that can affect the expression of the protein of interest. For example, a promoter / enhancer, suppressor, or exogenous transcription modulating element is inserted into the genome of the host cell in an orientation and proximity that are sufficient to influence the transcription of DNA encoding the desired truncated δΤΝΕΒ. Control elements do not encode truncated δΤΝΕΚ, but control a specific part of the DNA of the host cell genome. Thus, the expression of truncated δΤΝΕΚ. can be achieved not by transfection of the DNA encoding it, but by using targeting DNA (containing homology regions with the endogenous gene of interest) in combination with DNA regulatory elements that provide the endogenous gene sequence with recognizable transcription signals truncated δΤΝΕΚ

Cultivation of host cells.

Methods for culturing recombinant host cells to produce the desired protein will vary depending on many factors and conditions; the optimal procedure in each specific situation becomes clear to a qualified specialist after short experiments. Recombinant host cells are cultured in a suitable medium and expressed truncated δΤΝΕΚ. they are harvested, isolated, and purified from the culture medium (or from cells if it is expressed intracellularly) using methods known in the art.

Specifically, any recombinant cells used to produce truncated κΤΝΡΚ can be cultured in a medium that provides induction of promoters, selection of appropriate host cells, or amplification of a gene encoding the desired truncated κΤΝΡΚ. If necessary, hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate salts), buffers (such as HEPE8), nucleosides ( such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (inorganic compounds, the final concentration of which does not exceed micromoles), as well as glucose or other energy source. Other additives may also be administered in appropriate concentrations, which is obvious to a skilled person. Suitable culture conditions for particular host cells, such as temperature, pH, etc., are also well known to those skilled in the art.

The resulting expression product can be cleaned to almost homogeneity using methods known in the art. Examples of cleaning methods are described in applications EP 393438 and EP 422339, referred to in the present description by reference.

Pharmaceutical Compositions

Pharmaceutical compositions typically include therapeutically effective amounts of truncated κΤΝΡΚ and chemically modified truncated κΤΝΡΚ (combined by the term truncated κΤΝΡΚ product (s)) in admixture with a carrier. The carrier typically includes one or more pharmaceutically and physiologically acceptable materials in admixture with the truncated κΤΝΡΚ product (s) and controlled release material.

The primary solvent in the carrier can be either aqueous or non-aqueous in nature. In addition, the carrier may contain other pharmaceutically acceptable materials for modifying or maintaining the pH, preferably in the range of 5-6.5 and more preferably in the range of 5.5-6.0 (for example, buffers such as nitrate, phosphate, and such amino acids, like glycine); fillers for lyophilized forms (e.g. mannitol or glycine); substances for maintaining osmolarity (for example, mannitol or sodium chloride); surfactants (e.g., polysorbate 20, polysorbate 80, triton and P1igoshsk); substances to maintain viscosity, transparency, color, sterility, stability (for example, sucrose or sorbitol); antioxidants (e.g. sodium sulfite and sodium hydroxysulfite);

preservatives (e.g., benzoyl acid and salicylic acid); substances for preserving the smell of the pharmaceutical form; flavors and solvents; substances that determine the rate of dissolution (for example, solubilizers or solubilizing agents such as alcohols, polyethylene glycols and sodium chloride); substances that determine the rate of release; emulsifying agents; suspending agents; solvents; fillers; delivery vectors; diluents and / or pharmaceutical adjuvants. Effective administration forms such as slow release parenteral formulations, inhaled aerosols, oral formulations, or suppositories are also contemplated. Compositions may also include specific formulations of polymeric compounds, such as mass-degradable polymers (eg, poly (lactocoglycolic acid) copolymers (POA), polymer blends POA, block copolymers of PEOA and lactic and glycolic acid, polycyanoacrylates); surface-degradable polymers (e.g., polyorthoesters polyanhydrides); hydrogel esters (e.g., pluronic polyols, poly (vinyl alcohol), poly (vinyl pyrrolidone), copolymers of maleic anhydride and alkyl vinyl ether, cellulose, hyaluronic acid derivatives, alginate, collagen, gelatin, albumin, starches and dextrans and systems) liposomes or microspheres. Such compositions may affect the physical condition, stability, release rate ίη νίνο and clearance rate ίη νίνο of these proteins and their derivatives. The optimal pharmaceutical composition of a particular protein is determined by a qualified specialist, depending on the route of administration and the desired dosage. Examples of pharmaceutical compositions are described in Keshtop'k P11agtaseibs1 8s1epsek, 18 ' ¹ ' Eb. (1990, Musk RnMppd Co., EakYup, RA18042). p. 1435-1712; Otlokh apb Rey (1995), Vyusopscda1e Syet., 6: 332-351; Leopewow, e! A1., (1995), 1Oigpa1 OG Meb1 USA1 SketMgu, 38: 4263-4269; Naak e! A1., (1995), С11шса1 Ιιιιιιιιιι1 according to the app. \ UO 94/06457; \\ Ό 94/21275; ΡΚ 2706772 and \\ Ό 94/21235, referred to in the present description by reference.

Specific sustained release formulations are available from the following suppliers: Oero1ec11 (EeroGoat ™ ™, multivesicular liposomes); A1kegtek (PrigoEa ™ PbOA microspheres). As follows from the context of the present description, hyaluronan is understood to mean hyaluronan, hyaluronic acid, its salts (such as sodium hyaluronate), esters, enzymatic derivatives, and crosslinked hyaluronic acid gels (such as gilan). Examples of the forms of hyaluronan are described by Reugop apb Ba1axk (1974), RaSh Vyu., 22 (8), 731-736; Nba1e e! A1., (1991), GP Guide Weu., 4 (2): 93-99; Tag e! A1., (1993), 1opgpa1 oG Vyuteb1sa1 Ma! epak Kekeagsy, 27: 1129-1134; Yat1k1 e! A1., (1982), Sh! eta yupa1 Shita1 oG C11shsa1 Ryagtasodu, Luger apb ^ x ^^ du, 20 (11): 501-507; Meueg e! A1., (1995), Shita1 oG Sop! go11eb Ke1eake, 35: 67-72; K1kisy e! A1., (1996), Ok! eoagShgShk apb SagShade, 4: 99-110; Buckeye e! A1., (1994), C11shsa1 OgShoreebkk apb Ke1a! eb Kekeagsy, 299: 282-292; Meuegk apb vgapb! (1995), 22 (9): 1732-1739; Baigep! e! A1., (1995), Ac! a Og11yur. BSAPB. 66 (266): 116-120; Saxope e! A1., (1995), Vyuta! epa1k, 16 (7): 569-574;

UegakyaShp e! A1., (1994), AgsUyek oG VusyetMgu apb Vyuryukkk, 313 (2): 267-273: VegpaYu e! A1., (1993), 1opgpa1 oG Vyutebka1 Ma! epaq Kekeags, 27 (5): 677-681: Τaap e! A1., (1990), Aik1gaNap1Oigpa1 oG Vu! esbodoodu, 4 (1): 38-43; Otlokh apb Re! Sh (1995), Vyuso) id1e Sb., 6: 332-351; in US Patents Yok.

4,636,524, 4,713,448,

4,772,419, 4.851,521,

4,582,865,4,605,691

4,716,154, 4,716,224,

4,957,774, 4,863,907,

5,128,326, 5,202,431, 5,336,767, 5,356,883; in applications for the European patent Yok. 0507604 A2, 0718312 A2 and \ UO 96/05845, referred to in the present description by reference. Specific hyaluronan compositions are available from the following suppliers: VuMa! Pkh, Shs. K1bdeye1b, Ν6 (Bupu1ks ™, a mixture of 90:10 liquid gilan and gilan gel); ΡΙ6ιπ B.R.A. Alapo Τе ^ te. YaGu Ηua1dap ™, sodium salt of hyaluronic acid from the cocks ridge (MV from ~ 500,000 to ~ 700,000); Kakep Ryagtaseibsa1 Co., B! B., ^ Kuo, 1arap Ar1g ™, 1% solution of hyaluronic acid from the cockscomb, (MV -700,000); Й аг та АВ о,,, Б Б о ск Ь 1 1,, Б еб еб еп ^ Ц ™ ™ ™,, cockerel hyaluronic acid, (MV ~ 4x10 ⁴ ); Oephute Sogrogaiop, Satbpde, MA (Bigdy soa! ™, recombinant hyaluronic acid); . Rgopoua V1oro1uteg, Ric, RoyktoiSh, ΝΗ high molecular weight (MW - 1,5-2,2h10 ⁶⁾ hyaluronic acid ΡΟΗ, obtained from cultures of B (Ger osossik hooer | betkik; MW sodium hyaluronate (MW ~ 1,0-1,! 6x10 ⁶ ) and sodium hyaluronate LU (MV ~ 1.5-2.2x10 ⁶ ); Ca1;

Lai! Epidep, B \\ 'Pheg1apb (hyaluronic acid, sodium salt, (company catalog for 1997, no. 385908), obtained from B! Ger! Ossosski kr.); ! П п п Сот Сот Сот ра ра,,,, с с с Ь,,, ΝΥ hyaluronic acid from the cocks ridge, (MV> 1x10 ⁶ ); Vyukup SH Shs., Sysado, Sh; Atexo1 Comp., Ebkop, Ν6 and Kuo ^ a Η like Koduo Co., B! B.,% Kuo, Shrap.

After preparation, the pharmaceutical compositions may be stored in sterile bottles in the form of a solution, suspension, gel, emulsion, solid or dehydrated or lyophilized powder. Such compositions may be stored in ready-to-use forms or forms requiring dissolution before administration (e.g., lyophilized).

One of the specific embodiments of the invention relates to kits necessary to obtain a single dose of the drug. Each kit may include a first container with dried protein and a second container containing an aqueous composition. The scope of the present invention includes such kits, which include single or multi-chamber prefilled syringes; such prefilled syringes (for example, liquid syringes and syringes such as LUO-1EC! ®, two-chamber prefilled syringes) are available from Wueig OTZ Kauepkigd, Oegtapu.

Application.

Truncated CTO products may be useful as research reagents, as well as therapeutic and diagnostic agents. The indicated truncated KLUKk can be used in the diagnostic tests Ш У1! Go and / or Ш у1уо to determine the amount of native CTL-! or 5ΪΝΡΚ-ΙΙ in tissue and organ samples or for the determination and / or isolation of cells that express ΤΝΡ (BsShop e! a1., (1995), Kirg). When testing tissues and organs, the radioactivity from ΤΝΡ 'A-truncated KL ^ Kk bound will be less when compared with the standardized binding curve of Ά-truncated ILC due to the binding of unlabeled native ^ ΚΡ ^ -Ι or ^ ΝΈΕ-ΙΙ to ΤΝΡ. Similarly, Ά-truncated can be used to detect the presence of ΤΝΡ in cells of various types.

The present invention also provides for the use of truncated QCNT products to produce antibodies and the use of these antibodies (in particular those that bind native QCJC or 5ΪΝΡΚ-ΙΙ). You can get antibodies that bind truncated for example, antibodies to epitopes of the amino acid sequence K [Suk ^19- Suk ¹⁰³ ] -K2 or to epitopes of the amino acid sequence K4- [Suk ^32- Suk ¹¹⁵ ] -Y5. A qualified specialist can apply the well-known published methods to obtain monoclonal and polyclonal antibodies or recombinant antibodies that specifically recognize and bind various proteins encoded by the amino acid sequences of the present invention. Similar antibodies can be used to purify and characterize a full-sized, mature 30 kD inhibitor ΤΝΡ and a full-sized, mature 40 kD inhibitor ΤΝΡ.

The present invention also relates to methods for treating specific diseases and clinical conditions (many of which can be characterized as inflammatory diseases), which are mediated by ΤΝΡ. A disease or clinical condition is considered an ΤΝΕ-caused disease if a spontaneous or experimental disease is associated with an increased content of ΤΝΕ in body fluids or in tissues adjacent to the focus of the disease. ΤΝΕ-caused diseases can also be recognized by the following two conditions: (1) pathological manifestations associated with the disease can be reproduced experimentally in animals by administration of ΤΝΕ and (2) the pathology induced in animal models of the disease can be suppressed or canceled by treatment with agents that inhibit the action of ΤΝΕ. Many of the ΤΝΕ-caused diseases satisfy two of the three conditions listed, while others satisfy all three conditions. An incomplete list of ΤΝΕ-caused diseases, as well as related manifestations and symptoms that can be treated according to the methods of the present invention, include adult respiratory distress syndrome; cachexia / anorexia; cancer (e.g. leukemia); chronic fatigue syndrome; graft versus host reaction; hyperalgesia; inflammatory bowel disease; neuroinflammatory diseases; ischemic / reperfusion injuries, including cerebral ischemia (brain damage due to trauma, epilepsy, hemorrhage or stroke, each of these factors can lead to neurodegeneration); diabetes (e.g. juvenile type 1 diabetes); multiple sclerosis; eye diseases; pain; pancreatitis pulmonary fibrosis; rheumatic diseases (e.g. rheumatoid arthritis, osteoarthritis, juvenile (rheumatoid) arthritis, seronegative polyarthritis, ankylosing spondylitis, Reiter's syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, systemic sclerosis, sclerosis, sclerosis, , rheumatic fever, polychondritis and polymyalgia rheumatica and polyarteritis of giant cells); septic shock; side effects of radiotherapy; systemic lupus erythematosus; temporary disease of the mandibular joint; thyroiditis and tissue transplantation.

Each truncated κΤΝΕΒ product can be administered to patients in therapeutically effective amounts for the treatment of “conditional diseases, as defined above, including acute and chronic inflammations such as rheumatic diseases (eg, Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, and staphylocockin-induced (septic) arthritis). The term patient, along with humans, includes animals (for example, cats, dogs and horses).

The truncated κΤΝΕΒ product can be used by topical, enteral or parenteral administration, including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intrathecal, intraarticular, articular intraventricular and intrathoracic injection or infusion. The truncated κΤΝΕΒ product can also be administered orally or through the mucous membrane, for example, intranasally, sublingually, cheek or rectally for systemic delivery.

It is preferable to administer truncated κΤΝΕΒ products by intraarticular, subcutaneous, intramuscular or intravenous injection. In addition, the truncated κΤΝΕΒ product can be administered by a continuous infusion method (for example, using implanted or external devices providing a constant or intermittent supply of the drug) so as to constantly provide the desired level of the truncated κΤΝΕΒ product in the blood during administration. This is achievable, for example, when using various mini-pumps, such as an osmotic mini-pump. With this method of administration, you can be sure that the amount of the drug is maintained at the right level, you can take blood samples and control the amount of the drug in the bloodstream. Various pumps are commercially available from suppliers such as М1шМеб 1пс., 8у1шаг, SA (for example, МТ507) and А1ха Сгр., Pa1o Ayo, SA (for example, Aye osmotic pump !, 2MY model).

It is also believed that other methods of constant or almost constant delivery can be applied. For example, chemical derivatization can lead to the creation of sustained release forms of the protein, which affects the constant presence of the drug in the bloodstream in predictable amounts, moreover, this prediction is based on the dosage schedule.

Methods of using truncated κΤΝΕΒ products for the treatment of ΤΝΕ-related diseases, including inflammatory joint diseases (e.g., osteoarthritis, psoriatic arthritis and rheumatoid arthritis), are described in European Patent Application 567566, incorporated herein by reference. According to one specific embodiment of the invention, truncated κΤΝΕΒ products can be administered intraarticularly for the treatment of rheumatoid arthritis and osteoarthritis. According to another specific embodiment, truncated κΤΝΕΒ products can be administered subcutaneously or intramuscularly to treat rheumatoid arthritis, inflammatory bowel disease, cachexia / anorexia, or multiple sclerosis. According to another specific embodiment, truncated δΤΝΓΤΝ products. can be administered intravenously to treat brain damage due to trauma, epilepsy, hemorrhage or stroke, or administered intraventricularly to treat brain damage due to trauma. A preferred method for treating arthritis includes: (1) a single intraarticular injection of a truncated δΤΝΕΚ product, administered periodically to prevent or treat an arthritis attack, and (2) periodic subcutaneous injections of a truncated δΤΝΓΚ product. Treatment of septic shock should begin as soon as possible after septicemia or diagnosis of the possibility septicemia. For example, treatment may begin immediately after a surgery or an accident, or in the event of any event involving a risk of septic shock. Preferred methods for treating adult respiratory distress syndrome include: (1) single or multiple intratracheal administration of a truncated δΤΝΓΚ product. and (2) bolus or continuous intravenous infusion of truncated δΤΝΓΚ .. product

Another aspect of the invention relates to cell therapy, i.e. implantation of truncated δΤΝΓΚ producing cells. This embodiment provides patients with implantation of cells capable of synthesizing and secreting the biologically active truncated δΤΝΓΚ form. Such cells may be cells that normally do not produce truncated δΤΝΓΚ., but have been modified to produce truncated δΤΝΡΚΓΚ. , or cells whose ability to produce truncated δΤΝΓΚ. was improved by transformation with a polynucleotide suitable for expression and secretion of truncated δΤΝΡΚ

To minimize the possible immunological response of the patient to the introduction of truncated δΤΝΓΚ. of another species, it is preferable that the source cells are of human origin, or that the cells are encapsulated in a material that provides protection against immune recognition, or that the cells are placed in immunologically privileged areas, such as testicles, the eye, or the central nervous system.

Human or animal cells can be implanted into the patient in biocompatible semipermeable polymer capsules or membranes that allow the release of truncated δΤΝΡΚ, but prevent cell destruction by the patient’s immune system or other adverse factors from the surrounding tissues. Alternatively, the patient’s own cells can be transformed ex | yo to produce truncated 5–5 and then implanted into the patient without encapsulation. Methods of membrane encapsulation of living cells are known to qualified specialists and therefore the preparation of encapsulated cells and their implantation into patients may well be carried out.

As one of the embodiments of the invention, gene therapy 1p y | oo is considered when the nucleotide sequence encoding truncated δΤΝΕΚ is introduced directly to the patient. For example, encoding a truncated δΤΝΓΚ. the nucleotide sequence can be introduced into target cells by local injection of the nucleotide construct with or without an appropriate delivery vector. For example, encoding a truncated δΤΝΓΚ. the nucleotide sequence can be inserted into a vector based on an adeno-associated virus. Alternative viral vectors include, but are not limited to, vectors based on retroviruses, adenoviruses, herpes simplex virus, and papilloma virus. Physical transfer of both yyyyy and exyyyyyyo is also acceptable and can be accomplished by liposomal transfer, direct injection (naked DNA), receptor-mediated transfer (ligand-DNA complex) or microparticle bombardment (gene gun).

Examples of cell and gene therapy methods are described in US Pat. No. 4,892,538, US Pat. No. 5,011,472; U.S. Patent No. 5,106,627; No. 4219626, AO 94/20517 and 96/22793, referred to in the present description by reference.

Regardless of the route of administration, the treatment of ΤΝΕ-caused diseases requires doses or truncated δΤΝΡΚ dosage regimens effective to reduce or eliminate the symptoms of the disease. Other factors on which the appropriate dosage depends include the type of disease or clinical condition that needs to be treated or prevented, the severity of the disease, route of administration, age, gender, and clinical condition of the patient. Subsequent calculations necessary to clarify the appropriate dosage can be easily performed by a qualified specialist, especially taking into account the specific information provided in the present description. Dosage can also be determined using known dosage tests used in combination with appropriate dose-response data.

The frequency of administration depends on the pharmacokinetic parameters of truncated δΤΝΓΚ. in the pharmacological forms used. Truncated δΤΝΓΚ. can be administered once or in the case of acute and prolonged disorders, administered daily in smaller doses, or administered as an initial bolus dose, followed by a constant dose or sustained release. With parenteral administration, a single parenteral dose, for example, can reach 10 mg, usually up to 15 mg, and more preferably up to 20 mg. When introduced into the joint cavity, the pharmaceutical composition is preferably administered as a single injection, for example, from 3 to 10 ml or from 5 to 10 mg / ml of truncated δΤΝΕΚ dissolved in isotonic phosphate buffered saline. The drug can be injected into the joint cavity with a frequency, for example, once every 7-10 days. With this scheme, the introduction is carried out continuously, for example, 4-5 times, changing the dose if necessary.

In some cases, the administration of truncated δΤΝΕΚ products may form part of combination therapy in conjunction with other pharmaceutical compositions indicated for the treatment of the disease. The product of truncated δΤΝΕΚ and one or more traditional or new anti-inflammatory drugs can be administered separately or in combination.

Truncated δΤΝΕΚ products (for example, K ₁ - [Su8 ¹⁹ -Cu§ ¹⁰³ ] -K ₂ proteins) and any one or more additional anti-inflammatory drugs can be administered separately or in combination. Information on these compounds can be found in the Τίκ Megk Mapia1 o £ O | adpo515 apb Τye ^ aru guide. 81х1ееп1й ЕБШоп, Мегск, 8Наг & ЭоНше Кееэсгсь ЛаЬога1опе5, Мегск & Со., Кайгау, Ν1 (1992) and in РНагтаго) ес15, Р1В Рійсабопк Нб.

Existing treatment for ΤΝΕ-related diseases, as defined above, including acute and chronic rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcus-induced (septic) arthritis), primarily involves the use of a range of pain and inflammation control drugs classified as non-steroidal anti-inflammatory drugs (ΝδΑΙΌδ). The second tier of drugs includes corticosteroids, slow-acting anti-rheumatic drugs (δΑΑΡΌδ) or disease modifying drugs (MI).

In accordance with a specific embodiment of the invention, there is provided a method of using a truncated δΤΝΕΚ product (for example, protein K ₁ - [Su8 ¹⁹ -Su ¹⁰³ ] -K ₂ ) in combination (pre-treatment, post-processing or competitive processing) with one or more ΝδΝδ for treating ΤΝΕ -conditioned diseases as defined above, including acute and chronic rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococci-induced (septic) arthritis t) and the disease is graft versus host disease. ΝδΑΙΌδ exert their anti-inflammatory effect, at least in part, by suppressing the synthesis of prostaglandins (Soobtap apb Obtayt Τίκ P11agtasoodioca1 Bay5 o £ Τye ^ aireiί ^ s, MsMShap, 7111 EbIgop (1985). ΝδΑΙΌδ can be divided into nine groups: (1) salicylic acid derivatives; (2) derivatives of propionic acid; (3) derivatives of acetic acid; (4) phenamic acid derivatives; (5) carboxylic acid derivatives; (6) derivatives of butyric acid; (7) oxicams; (8) pyrazoles; and (9) pyrazolones.

According to a specific embodiment of the invention, there is provided a method of using a truncated δΤΝΕΚ product (for example, protein K ₁ - [Su8 ¹⁹ -Cu§ ¹⁰³ ] -K ₂ ) in combination (pre-treatment, post-treatment or competitive processing) with one or more salicylic acid derivatives, pre-drug esters or their pharmaceutically acceptable salts. Derivatives of salicylic acid, esters of drugs and their pharmaceutically acceptable salts include: acetaminosalol, aloxiprine, aspirin, benorilate, bromosaligenin, calcium acetylsalicylate, choline magnesium trisalicylate, difluzinal, etersalate, fendosal, gentizyl aminosalicin malisulfide, gentizyl aminisalic aminosalicyl aminisalic aminosalicyl aminisalic aminosalicyl aminosalicyl aminisalic aminosalicyl aminic aminosalicyl aminosalicin salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, oxyacetic acid salicylamide, salsalate and sulfasalazine. Structurally related derivatives of salicylic acid having similar analgesic and anti-inflammatory properties are also included in this group.

According to a specific embodiment of the invention, there is provided a method of using a truncated δΤΝΕΚ product (for example, protein K ₁ - [Su8 ¹⁹ -Cu§ ¹⁰³ ] -K ₂ ) in combination (pre-treatment, post-processing or competitive processing) with one or more derivatives of propionic acid, pre-drug esters or their pharmaceutically acceptable salts. Derivatives of propionic acid, esters of pre-drugs and their pharmaceutically acceptable salts include: alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen, phenoprofen, flunoxaprofen, flurbiprofen, furcloprofenoprofenoprofenoprofenoprofenoprofenoprofenoprofenoprofenoprofenopromenoprofenopromenoprofenoprofenoprofenoproprofenoproprofenoproprofenoproenoprofenoprofenoprofenoproprofenoproprofenoproenoprofenoprofenoprofenoproen , picketoprofen, pimeprofen, pirprofen, pranoprofen, protisinic acid, pyridoxyprofen, suprofen, thiaprofenic acid and thioxaprofen. Structurally related propionic acid derivatives having similar analgesic and anti-inflammatory properties are also included in this group.

According to a specific embodiment of the invention, there is provided a method of using a truncated δΤΝΕΚ product (for example, protein Κ ₁ - | ί \ δ ¹⁹ -ί.'νδ ^1ο3 | -Κ _; ) in combination (pre-treatment, post-processing or competitive processing) with one or more acetic derivatives acids, pre-drug esters or their pharmaceutically acceptable salts. Acetic acid derivatives, pre-drug esters and their pharmaceutically acceptable salts include: acetamycin, alklofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin, diclofenac sodium, ethodolac, felbinac, phenclofenacen, phenclacfenacin, phenclofenacenic acid, phenclofenacenic acid indomethacin, isofezolac, isoxipack, lonazolac, methiazinic acid, oxametacin, oxspinac, pimetacin, proglumethacin, sulindac, talmetacin, tiaramide, thiopinac, tolmetin, zidomethacin and zomepirac. Structurally related derivatives of acetic acid having similar analgesic and anti-inflammatory properties are also included in this group.

According to a specific embodiment of the invention, there is provided a method of using a truncated δΤΝΡΚ product (for example, protein Κ ₁ - | ί \ δ ¹⁹ -ί.'νδ ^1ο3 | -Κ _; ) in combination (pre-treatment, post-processing or competitive processing) with one or more phenamine derivatives acids, pre-drug esters or their pharmaceutically acceptable salts. Derivatives of fenamic acid, esters of pre-drugs and their pharmaceutically acceptable salts include enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamate sodium, medofenamic acid, mefanamic acid, nifluminic acid, tolfenamenamate, tolnenfenamate, tolnenfenamate, tolnenfenamate, tolnenfenamate, tolnenfenamate, tolnenfenamate, tolnenfenamate, tolnenfenamate, tolnifenamate, tolnenfenamate, tolfenamate; Structurally related phenamic acid derivatives having similar analgesic and anti-inflammatory properties are also included in this group.

According to another embodiment of the invention, there is provided a method of using a truncated δΤΝΡΚ product (for example, protein Κ ₁ - | ί \ δ ¹⁹ -ί.'νδ ^1ο3 |-в _; ) in combination (pre-treatment, post-processing or competitive processing) with one or more carboxylic derivatives acids, pre-drug esters or their pharmaceutically acceptable salts. Carboxylic acid derivatives, pre-drug esters and their pharmaceutically acceptable salts include: clidanac, diflunisal, flufenizal, inoridine, ketorolac and tinoridine. Structurally related carboxylic acid derivatives having similar analgesic and anti-inflammatory properties are also included in this group.

According to another embodiment of the invention, a method for using a truncated δΤΝΡΚ product (for example, protein белка ₁ - | ί \ δ ¹⁹ -ί.'νδ ^1ο3 | -Κ _; ) in combination (pre-treatment, post-processing or competitive processing) with one or more derivatives of butyric acid, esters of the drugs or their pharmaceutically acceptable salts. Derivatives of butyric acid, esters of the drugs and their pharmaceutically acceptable salts include boomdisone, butibufen, fenbufen and xenbucin. Structurally related derivatives of butyric acid having similar analgesic and anti-inflammatory properties are also included in this group.

According to another embodiment of the invention, there is provided a method of using a truncated δΤΝΡΚ product (for example, protein | ₁ - | ί \ δ ¹⁹ -ί.'νδ ^1ο3 | -Κ _; ) in combination (pre-treatment, post-processing or competitive processing) with one or more oxycams, esters of the drugs or their pharmaceutically acceptable salts. Oxycams, esters of drug preparations and their pharmaceutically acceptable salts include droxics, enolics, isoxics, piroxicam, sudoxic, tenoxic and 4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4- (Ν phenyl) -carboxamide. Structurally related oxycams having similar analgesic and anti-inflammatory properties are also included in this group.

In accordance with another embodiment of the invention, there is provided a method of using a truncated δΤΝΡΚ product (for example, Κ-Κνδ ¹⁹ -ί.'νδ ^1ο3 |-белка protein _; in combination (pre-treatment, post-processing or competitive treatment) with one or more pyrazoles, pre-drug esters or their pharmaceutically acceptable salts. Pyrazoles, esters of drug preparations and their pharmaceutically acceptable salts include diphenamisole and epirisole. Structurally related pyrazoles having similar analgesic and anti-inflammatory properties also include are in this group.

In accordance with another embodiment of the invention, there is provided a method of using a truncated δ продукта product (for example, Κ-Ννδ ¹⁹ -ί.'νδ ^1ο3 | -Κ _; protein) in combination (pre-treatment, post-processing or competitive processing) with one or more pyrazolones, pre-drug esters or their pharmaceutically acceptable salts. Pyrazolones, drug esters and their pharmaceutically acceptable salts include apazone, azapropazone, benzpiperilone, feprazone, mofebutazone, torazone, oxyphenbutazone, phenylbutazone, pipebuzone, propylphenazone, ramiphenazone, suksibazone and tuxinbazone. Structurally related pyrazolones having similar analgesic and anti-inflammatory properties are also included in this group.

In accordance with another embodiment of the invention, there is provided a method of using a truncated δ например product (for example, protein Κ-Ννδ ¹⁹ -ί.'νδ ^1ο3 |-белка _; ) in combination (pre-treatment, post-processing or competitive treatment) with one or more of the following K / .AYUK: ε-acetamidocaproyl acid, 8-adenosinemethionine, 3-amino-4 hydroxybutyrylic acid, amiksetrin, anitrazafen, anthraphenin, bendazak, bendazak lysinate, benzydamine, beprosin, broperamol, bucol, bufesolac, ciprozide, ciprozide, ciprozide, ciprozide , difenpayr mid, diphenpiramide, diphysalamine, ditazole, emorphazone, fanethylisole mesylate, fenflumisole, flactaphenin, flumizole, flunixin, fluproquazone, fopyrtoline, phosphozal, quimesinfismolinoliznolisolnolizolnoliznolizolnomoliznolizolnizlin , nimesulide, orgotein, opranoxin, oxacetrolm, oxapadol, paraniline, perisoxal, perisoxal citrate, pifoxime, piproxen, pyrazolac, pirfenidone, proquazone, proxazole, thielavin B, tiflamisole, thimegadine, toltin, toltipt, toltip, toltip, toltip other company codes as 4801568, AA861, ΆΌ1590,

AER802, AER860, ΑΙ77Β, AP504, AI8001, VRRS, V \ 540C, SNGM0GN 127, SM00, EV382, Е508, Е1044, ЕК-506, 6У3658, ΠΈ182, КСкТЕК090, КМЕ4, ЛА2851, MV714,

0 ^ 3144, РВ823, РУ102, РУ108, В830, В82131, 8СВ152, 8Н440, 8ΙΒ133, 8RA8510, 8Р27239, 8Т281, 8Υ6001, TA60, TAE901, 4-benzoyl-1indanecarboxylic acid, ТУХ2707,

I60257, IV2301 and Υ41770. Structurally related ZHJUK having similar analgesic and anti-inflammatory properties are also included in this group.

In accordance with one embodiment of the invention, a method of using a truncated kT # K product is provided. (for example, protein B ₁ - [Suk ^19- Suk ¹⁰³ ] -B ₂ ) in combination (pre-treatment, post-processing or competitive processing) with one or more corticosteroids, esters of the drugs or their pharmaceutically acceptable salts for the treatment of T№-caused diseases, such as defined above, including acute and chronic rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcus-induced (septic) arthritis) and multiple sclerosis. Hydrocortisone and compounds derived from hydrocortisone, such as 21-acetopregnenolone, alklomerazone, algestone, amcinonide, beclomethasone, betamethasone, betamethasone valerate, budenisonide, chloropetazole clonazetazole, clopazetazole, clopazetazole, clozetazole butyrate, clocortocolon, cloprednol, corticosterone, cortisone, cortivazole, deflazacone, desonide, deoxymerazone, dexamethasone, diflorazone, diflucortolone, difluprednat, enoxolone, fluazacort, fluc SPEAR, flumethasone, flumethasone pivalate, flunisolide, flucinolone acetonide, fluocinonide, fluorotsinolon acetonide, fluorokortin butyl, fluorokortolon, fluorokortolon hexanoate, diflucortolone valerate, fluorometolon, fluperolon acetate, Fluprednidene acetate, fluprednisolone, flurandenolid, formocortal, galtsinonid, halometasone, halopredone acetate, gidrokortamat , hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate, hydrocortisone tebutate, mazipredon, medrisone, meprednisone, methylpredn col, mometasone furoate, parametasone, predicarbate, prednisolone, prednisolone 21-dihedriaminoacetate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone sodium 21-tsulfobenzoate, prednisolone sodium 21, stearoglycolate, prednisolone acetate, prednisolone acetate, prednisolone acetate, prednisolone acetate , thixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide and triamcinolone hexacetonide. Structurally related corticosteroids having similar analgesic and anti-inflammatory properties are also included in this group.

According to another specific embodiment of the invention, there is provided a method of using a truncated CTXER product (for example, protein B1- [Suk ^19- Suk ¹⁰³ ] -B ₂ ) in combination (pre-treatment, post-processing or competitive processing) with one or more slowly acting anti-rheumatic drugs (8 AAAEC) or disease-modifying antirheumatic drugs (OMAKEc), pre-drug esters or their pharmaceutically acceptable salts for the treatment of T№-caused diseases, as defined above, including acute and onicheskie rheumatic diseases (e.g., lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and stafilokokkindutsirovanny (septic) arthritis) and multiple sclerosis. 8AAKEk or OMAKEk, pre-drug esters and their pharmaceutically acceptable salts include: allocupreide sodium, auranofin, aurothioglucose, aurothioglucanide, azathioprine, sodium brequinar, butylamine, calcium 3-aurotio-2-propanol-chloroquinoloculfonocarcinol-1-culfonocarboxene-sulfonocarboxene-sulfonocarboxene-sulfonocarboxene-sulfonocarboxene-sulfonocarboxene-sulfonocarboxene , cyclophosphamide, cyclosporine, dapzone, 15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., gold salt of cycloquin, gold salt of thiomalate, gold salt of thiosulfate), hydroxychloroquine, hydroxyurea, kebuzon, levamisole, lobenzarite, erkaptopurin, methotrexate, mizoribine, mycophenolate mofetil, marjoram, mustard, nitrogen, Ό-pennitsillamin such pyridinol imidazoles as 8KHE86002 and 8V203580, brine mitsin, thiols, thymopoietin and vinkrestin. Structurally related 8AAVEc or OMAVEK having similar analgesic and anti-inflammatory properties are also included in this group.

In accordance with another specific embodiment of the invention, there is provided a method of using a truncated CTJ product (for example, protein B1- [Suk ^19- Suk ¹⁰³ ] -B ₂ ) in combination (pre-treatment, post-processing or competitive treatment) with one or more COX2 inhibitors, pre-drug esters or their pharmaceutically acceptable salts for the treatment of ΤΝΡ-related diseases, as defined above, including acute and chronic inflammation. An example of COX2 inhibitors, drug esters or their pharmaceutically acceptable salts is, for example, celecoxib. Structurally related COX2 inhibitors having similar analgesic and anti-inflammatory properties are also included in this group.

In accordance with another specific embodiment of the invention, there is provided a method of using a truncated K продуктаNΡB product (for example, protein B ₁ - [Suk ^19- Suk ¹⁰³ ] -B ₂ ) in combination (pre-treatment, post-processing or competitive treatment) with one or more antimicrobial agents, esters drugs or their pharmaceutically acceptable salts for the treatment of ΤΝΡ-related diseases, as defined above, including acute and chronic inflammation. Antimicrobial agents include, for example, ampicillin, amoxicillin, aureomycin, bacitracin, ceftazidime, ceftriaxone, cefotaxime, cefachlor, cefalexin, cefradine, ciprofloxacin, clavulancylcylcyclincyllancyclincyclincyllancylancyllancylancylancylancylancylancyllidin penicillin and vancomycin. Structurally related antimicrobial agents having similar analgesic and anti-inflammatory properties are also included in this group.

In accordance with another specific embodiment of the invention, a method of using a truncated kNV product (for example, protein B ₁ - [Suk ^19- Suk ¹⁰³ ] -B ₂ ) in combination (pre-treatment, post-treatment or competitive treatment) with one or more of the following drugs for treatment ΤΝΡ conditional diseases as defined above, including acute and chronic inflammation: thalidomide; YOU 50730; tenadap; E 5531; thiapafant PCA 4248; nimesulide; Panavir rolipram; RV 73401; peptide T; ME 201; 449A; (1B, 38) -C1k-1- [9- (2,6-diaminopurinyl)] - 3hydroxy-4-cyclopentene hydrochloride; (1B, 3B) trans-1- (9-adenyl) -3-azidocyclopentane hydrochloride; and (1B, 3B) -trans-1- (6-hydroxypurin-9yl) -3-azidocyclopentane.

According to another specific embodiment of the invention, there is provided a method of using a truncated KIEV product (for example, protein B! - [Suk ^19- Suk ¹⁰³ ] -B ₂ ) in combination (pre-treatment, post-processing or competitive treatment) with one or more additional inhibitors ΤΝΡ for treatment ΤΝΡ -conditioned diseases, as defined above, including acute and chronic inflammation. Inhibitors of ΤΝΡ are compounds and proteins that block the synthesis of ΤΝΡ 1p y | yo and the release of ΤΝΡ from the cell.

Additional inhibitors of ΤΝΡ include anti-ΤΝΡ antibodies, for example, MAK 195 Ρ Ρ Ь antibodies (Ho11eg e! A1., (1993), 1k! Iegpaiopa1 8 troopst op Su! Window wopope Maggote Τ ^ apkr1apίa! ^ Op, 147; COP 571 anti -ΤΝΡ monoclonal antibodies (Vapksh e! A1., (1995), Vg1k11 1oigpa1 oG Vreita! Oodu, 34: 334-342; works are referred to in the present description by reference); mouse anti-monoclonal antibodies BAΥ X 1351 (Glue e ! a1., (1995), 7111 Eigoreap Sopdgekk oG S11shsa1 M1sgoOo1ogu apy IchGesyoik P1keakek, 9; the work is mentioned in the present description by reference); СеΤΤNΡ сА2 anti-ΤΝΡ monok personal antibodies (ESh e! a1., (1994), Laps! !, 344: 1125-1127 and ESh e! a1., (1994), Laps !, 344: 1105-1110; references are referred to in this description) .

In accordance with a particular embodiment of the invention, there is provided a method of using a truncated ^ ΧΡΕ product (for example, protein B1- [Suk ^19- Suk ¹⁰³ ] -B ₂ in combination (pre-treatment, post-processing or competitive processing) with soluble recombinant human antigen or its recombinant variants (AO 96/20206 and Mopp1x e! A1., Uttipodu, 155: 4829-4837; EP 510 681; publications are referred to in the present description by reference). In the application AO 96/20206 describes a secreted human antigen (natural and recombinant, including fusion protein), cn events for isolating genes responsible for encoding a soluble recombinant human antigen, methods for gene cloning in acceptable vectors and cell types, and gene expression methods for producing inhibitors EP 510691 describes DNAs encoding a human antigen, including soluble antigen, vectors for the expression of these DNA, and transformation cells transfected with the vector.With parenteral administration, the doses of fusion protein as antigen range from 1 to 100 μg / kg.

In accordance with a specific embodiment of the invention, there is provided a method of using a truncated kNV product (for example, protein B! - [Suk ^19- Suk ¹⁰³ ] -B ₂ ) in combination (pre-treatment, post-treatment or competitive treatment) with one or more interleukin-1 inhibitors for treatment of ленных caused diseases as defined above, including acute and chronic rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, and staphylococcus ny (septic) arthritis); brain damage due to trauma, epilepsy, hemorrhage or stroke, as well as multiple sclerosis. Interleukin-1 inhibitors include interleukin-1 receptor antagonists (any compound capable of specifically preventing the activation of GS-1 cellular receptors), such as IIIa, as described below; anti-W-Ereceptor antibodies (for example, EP 623674, referred to in the present description by reference); BS-1 binding proteins such as soluble III receptors (for example, US Pat. No. 5,492,88, US Pat. No. 5,488,032, US Pat. No. 5,464,937, US Pat. No. 5,319,071 and US Pat. No. 5,180,812, incorporated herein by reference); anti-HL monoclonal antibodies (for example, Ο 9501997, Ο 9402627, Ο 9006371, US patent 4935343, EP 364778, EP 267611 and EP 220063 referred to in the present description by reference); auxiliary proteins of the K-1 receptor. for example, \ O 96/23067 (referred to in the present description by reference) and other compounds and proteins that block the synthesis of w | yo or extracellular release of W-1.

Interleukin-1 receptor antagonist (Sh1ga) is a human protein that acts as a natural Sh-1 inhibitor. Preferred receptor antagonists, as well as methods for their preparation and methods for their use are described in US patent No. 5,075,222 (denoted as patent 222); \ O 91/08285; \ O 91/17184; Au 9173636; \ O 92/16221; \ O 93/21946; published international application No. i8 97/02131, which also describes a pharmaceutical composition comprising (a) an effective amount of a polymer for controlled release (eg, hyaluronic acid) and (b) an effective amount of ΙΌ-1 ha; \ About 94/06457; \ O 94/21275; ΡΚ 2706772; \ O 94/21235; ΌΕ 4219626, \ O 94/20517 and \ O 96/22793, referred to in the present description by reference. These proteins include both glycosylated and non-glycosylated Sh-1 receptor antagonists.

Specifically, the three preferred forms of IgA are (Sh-1gaa, Yu-1 τηβ and Ι0-1 gah). derived from a single DNA coding sequence are claimed and described in US Pat. No. 5,075,222, Yashit e! A1. entitled Interleukin-1 Inhibitors. This US patent, referred to as patent 222, is specifically incorporated into this description by reference. All three of these interleukin-1 inhibitors have similar functional and immunological activities.

Methods for producing inhibitors of interleukin1, in particular ELgax. also described in patent 222. One of the methods provides for the isolation of inhibitors from human monocytes (where they are normally produced). The second of the described methods involves the isolation of the gene responsible for coding ΙΌ-1 gas, the cloning of the gene in convenient vectors and cell types, the expression of the gene to produce SHLgas and the allocation of Sh-1 gas. The latter method, which is a special case of the use of recombinant DNA methods in general, is most preferred for the implementation of the present invention. According to a particular embodiment of the invention, Yu-1 ha contains a terminal methionyl group as a result of expression in E. soi. Modified S1gas are also within the scope of the present invention. Modified U-1 gas includes, for example, muteins of such inhibitors in which cysteine residues are replaced by another amino acid at one or more sites in the amino acid sequence of a natural inhibitor. It is possible to provide a site-specific reaction of such muteins with functionalized units of polyethylene glycol (PEC) or with other sulfhydryl-containing polyesters to obtain forms of HLGA-PEC. The published application \ O 92/16221 describes a number of modified forms of ELTA and methods for producing such RES-modified inhibitors.

An additional class of interleukin-1 inhibitors includes compounds that can specifically prevent the activation of cell receptors Sh-1. Such compounds include III-binding proteins, such as soluble receptors and monoclonal antibodies. These compounds also include monoclonal antibodies to receptors.

Another class of interleukin-1 inhibitors includes compounds and proteins that block the synthesis and / or extracellular release of S-1. Such compounds include agents that affect the transcription of EC-1 genes or the processing of ΙΕ-1 preproteins.

The above are examples that do not exclude other treatment options that can be used competitively with these anti-inflammatory compounds known to the skilled artisan, or can be developed by the skilled artisan taking into account the recommendations given in the present description.

It is particularly preferable to obtain compositions of additional anti-inflammatory compounds in unit dosage form to facilitate administration and constant dosage. The term unit dosage form refers to physically discrete units, each unit containing a predetermined amount of an additional anti-inflammatory compound, calculated to produce the desired therapeutic effect, in combination with the necessary pharmaceutical carrier. The term “pharmaceutically acceptable carrier” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption retarding agents and the like, which are compatible with the active ingredient, route of administration and other ingredients, as well as are not harmful to the recipient.

The use of such media and agents is well known in the art (see, for example, RettDiUp'k Rbagtasei! 1ca1 8aepsek, 18 ' ¹ ' Eb. (1990) Mask RiNkYpd Co., Eak! Op, RA 18042, pp. 1435-1712 ; the manual is incorporated herein by reference). Additional active ingredients may be included in the composition.

For oral therapeutic use, the additional anti-inflammatory compound can be mixed with excipients and used in the form of digestible tablets, cheek tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., or it can be administered directly into food. Tablets, lozenges, pills, capsules and the like dosage forms may also contain binders such as tragacanth gum, gum arabic, starch or gelatin; fillers such as dicalcium phosphate; disintegrating agents such as starch, alginic acid and the like; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; flavors such as peppermint, pear oil, cherry or orange flavors. When the unit dosage form is a capsule, it may contain, in addition to the agents listed above, a liquid carrier. Other materials may also be present that, in the form of a coating or otherwise, modify the physical form of a unit dose. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used to produce a unit dosage form must be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, an additional anti-inflammatory compound may be incorporated into the formulation or composition for sustained release. The amount of additional anti-inflammatory compound in such therapeutically useful compositions is such that the desired dosage is achieved.

For parenteral therapeutic use, each additional anti-inflammatory compound may be administered in a sterile injection solution. Sterile injectable solutions can be prepared by administering an additional anti-inflammatory compound in the required amount into an appropriate pharmaceutically acceptable carrier with various other ingredients listed below (if necessary), and then sterilized by filtration. Dispersions can be obtained by introducing an additional anti-inflammatory compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed above. If it is necessary to prepare sterile injectable solutions, they can be obtained by introducing a powder of an additional anti-inflammatory compound and optionally any other desired ingredient into a solution pre-sterilized by filtration, while the powder is prepared by any convenient method (for example, vacuum drying or lyophilization).

The specific dose of the additional anti-inflammatory drug is calculated based on the approximate body weight or body surface of the patient. Among the factors determining the appropriate dosage, there may be a specific nosological form that needs to be cured or prevented, the severity of the disease, the route of administration, as well as the age, gender and clinical condition of the patient. Subsequent refinement calculations necessary to determine the appropriate dosage are routinely performed by qualified specialists. Dosage may also be determined on the basis of known dosage tests in combination with appropriate dose-response data.

For example, the scope of the present invention includes dosages of additional anti-inflammatory compounds for treating specific acute and chronic inflammatory diseases such as rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, and staphylococcal-induced (septic arthritis), which may vary to achieve the desired therapeutic effect. If one of the additional anti-inflammatory compounds has side effects, it can be prescribed to patients during alternating periods of combination therapy. For example, chronic treatment with methotrexate is associated with toxicity to the gastrointestinal tract, liver, bone marrow, and lungs (8аηбονа1 е! А1., (1995), Vpekb 1oita1 oG Kbeita! O1odu, 34: 49-54;

the work is referred to in the present description by reference).

Methods of controlling the course of the disease may include specific tests aimed, for example, to determine a systemic response to inflammation, which include the erythrocyte sedimentation rate (E8K) and the presence of acute phase reactants (ARCs). The presence or absence of edema in the involved parts of the body is also noted. An increase in joint mobility and a decrease in pain in the patient are also noted. If the patient's condition is stable, he is prescribed the same dosage weekly and his condition is evaluated weekly. Given that the patient's condition is stable, treatment can be continued. After six months of treatment, the presence of anatomical changes in the skeleton is determined using radiological methods, for example, radiography.

At the end of each period, the patient's condition is re-evaluated. Comparison of radiological parameters, E8K and ARC before and after treatment indicates the effectiveness of treatment. Depending on the effectiveness of the treatment and the condition of the patient, the dosage may be increased or left unchanged for the entire duration of treatment.

Preferably, the present invention relates to a method of using one of the following combinations to treat or prevent an acute or chronic inflammatory disease as defined above, such as rheumatic diseases (e.g. Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, and staphylococci-induced (septic) arthritis), a product of truncated kT # K (for example, protein K ₁ [Suk ^19- Suk ¹⁰³ ] -K2) and methotrexate; a truncated CTTC product (for example, K1- [Suk ¹⁹ Suk ¹⁰³ ] -K2 protein), methotrexate and an inhibitor 1b-1, preferably 1b-1ha; a truncated CTTC product (for example, protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K2) and one or more of the following components: methotrexate, immunosuppressor (eg cyclosporin), ciprofloxacin, Eac antigen and ΙΙ-1 inhibitor, preferably 11-1 ga ; truncated cJTC product (for example, K1- [Suk ^19- Suk ¹⁰³ ] -K2 protein), methotrexate and immunosuppressant (for example, cyclosporin); the product of truncated kT№K (for example, protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K2), methotrexate and ciprofloxacin; a truncated CTTC product (for example, K1- [Suk ^19- Suk ¹⁰³ ] -K2 protein) methotrexate and a Ι1-1 inhibitor, preferably 11-1 ha; the product of truncated kT№K (for example, protein K ₁ - [Suk ¹⁹ Suk ¹⁰³ ] -K2) and one or more of the following components: methotrexate, sulfasazine and hydroxychloroquine; a truncated CTTC product (for example, K1- [Suk ^19- Suk ¹⁰³ ] -K2 protein), methotrexate and hydroxychloroquine; truncated CTTC product (for example, protein K ₁ - [Suk ¹⁹ Suk ¹⁰³ ] -K ₂ ), methotrexate sulfasazine.

According to a specific embodiment of the invention, there is provided a method of using (for example, intraarticular, subcutaneous or intramuscular injection) of a truncated CTJ product (for example, protein K ₁ - [Suk ¹⁹ Suk ¹⁰³ ] -K ₂ ) incorporated into a sustained release formulation (eg, hyaluronan ) additionally in combination (pre-treatment, post-treatment or competitive processing) with methotrexate and / or a Ι1-1 inhibitor (e.g. 11-1 ha) and / or soluble recombinant human EAC antigen for the treatment of rheumatic diseases, as defined but above (e.g., lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and Staphylococcus-induced (septic) arthritis) and the symptoms associated therewith.

According to another specific embodiment of the invention, there is provided a method of using (for example, intravenous or intraventricular administration) a truncated kT # K product (for example, protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K ₂ ) formulated for sustained release (for example, in hyaluronan) additionally in combination (pre-treatment, post-treatment or competitive treatment) with a tissue plasminogen activator and / or a Ι1-1 inhibitor (e.g. 11-1 ha) for the treatment of brain damage due to trauma, epilepsy, hemorrhage or stroke, When in use, each of these factors can lead to neurodegeneration.

According to another specific embodiment of the invention, there is provided a method of using (for example, subcutaneous or intramuscular injection) a truncated kT # K product (for example, protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K ₂ ) formulated for sustained release (for example, in hyaluronan) additionally in combination (pre-treatment, post-treatment or competitive processing) with one or more corticosteroids, cyclosporin, EK-506 or interferon (e.g. alpha interferon, beta interferon, gamma interferon or consensus interferon n) and / or 11-1ga for treating multiple sclerosis.

According to another specific embodiment of the invention, there is provided a method of using (for example, subcutaneous or intramuscular injection) a truncated kT # K product (for example, protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K ₂ ) formulated for sustained release (for example, in hyaluronan) additionally in combination (pre-treatment, post-treatment or competitive processing) with C-C8E and / or 11-1 g for the treatment of inflammatory bowel disease.

According to another specific embodiment of the invention, there is provided a method of using (for example, subcutaneous or intramuscular injection) a truncated κΤΝΡΚ product (for example, protein K ₁ - [Suk19-Suk ¹⁰³ ] -K ₂ ) incorporated into a sustained release formulation (eg, hyaluronan) additionally in combination (pre-treatment, post-treatment or competitive processing) with leptin, Mapio1 ™ or Medase ™ for the treatment of cachexia / anorexia.

According to another specific embodiment of the invention, there is provided a method of using (for example, subcutaneous, intraventricular or intrathecal administration) of a truncated κΤΝΕΚ product. (for example, protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K ₂ ), introduced into the composition for sustained release (for example, in hyaluronan) additionally in combination (pre-treatment, post-treatment or competitive treatment) with Ν8ΑΙΌ (for example, indomethacin) and / or an Ι1-1 inhibitor (e.g., P-1ga) for the treatment of Alzheimer's disease.

According to another specific embodiment of the invention, there is provided a method of using (for example, subcutaneous, intraventricular or intrathecal administration) of a truncated κΤΝΕΚ product. (for example, protein K ₁ - [Suk ^19- Suk ¹⁰³ ] -K ₂ ), introduced into the composition for sustained release (for example, in hyaluronan) additionally in combination (pre-treatment, post-treatment or competitive treatment) with soluble recombinant Cancer human antigen for treatment cancer (e.g., leukemia); diabetes (e.g. juvenile type 1 diabetes); graft versus host reactions; hepatitis A; ischemic reperfusion injuries, including cerebral ischemia (brain damage due to trauma, epilepsy, hemorrhage or stroke, each of these factors can lead to neurodegeneration); neuroinflammatory diseases; rheumatic diseases as defined above (for example, Lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcus-induced (septic) arthritis), as well as in tissue transplantation.

Other aspects and advantages of the present invention will be apparent upon consideration of the following illustrative examples.

Examples

Standard methods for carrying out many of the procedures described in the following examples, or acceptable alternative methods are given in well-known manuals on molecular biology, such as, for example, ebb e! A1., (1989), Kirg and AikBe1 e! A1., (1990), Kirg. For the convenience of the reader, ml stands for milliliters, and l stands for liters.

Example Ι.

The following example describes the preparation of various forms of truncated recombinant soluble κΤΝΕΚ.

ΝΗ ₂ -ΜΟ8ν0Ρ0ΚΥΙΗΡ0ΝΝ8ΐε- [0ν5 '' -Souz '° ⁵ ] -RS-COOH "ΓΝΡΚ-Ι 2.6О / С105);

ΝΗ _Γ ΜΟ5νεΡ0ΚΥΙΗΡ0ΝΝ5Ι0- [0γ5 ¹⁹ -Cy5 ^{, m} ] -PCB-COOH (δΤΝΡΚ-Ι 2.6P / C106);

UN ₂ -MO5USRUKU1NR0M№1S- [Su5 ^1E- Suz '^ LI-COOH (ϊΤΝΓΚ-Ι 2.6Ο / Ν105);

ON _{2 -} MU1NROY No. 1C- [Su2 ⁹ -Su5 ¹⁰⁵ ] -PCC8E-COOH (δΤΝΡΚ-Ι 2.3ϋ / ά8);

MH ₂ -M- [Su ₅ ¹⁹ -Su5 ^{| 05} ] -RYS5E-COOH (δΤΝΡΚ-Ι 2.3ϋ / ά18);

ΝΉ ₂ -Μ5Ι5- [Suz ¹⁹ -Su5 ^{, u} ] -RKS8E-COOH (δΤΝΡΚ-Ι 2.30 / 615).

Getting DNA.

1. κΤΝΡΚ-Ι 2.6B / S106.

PCR-Ι 2.6B / C106 PCR amplification was performed using the cloned cDNA obtained from the lambda-dr! 107c! HGp (EP422339) clone and the following PCR primers as the template:

5 ’HSE # 1: (8ΕΟ Yu ΝΟ: 68) 5'-OOTTAAOSSATATOOASASSO'GGTOSSSSSSA-3 '3' ΟΕΙΟΟ # 2: (8Εζ) Yu ΝΟ: 69) 5'-CCAAOOSTTTTASAOAOAOAOSAATTOAAOOSASTO-3’

OHYO # 1 and OHYO # 2 contained the restriction sites NbeI and Hshb and hybridized at the 5 'and 3' ends of the truncated gene, respectively. PCR amplification was performed for 25 cycles; each cycle consisted of 30 s at 94 ° С for denaturation, 15 s at 55 ° С for annealing, and 1 min at 72 ° С for elongation [model 2400 thermo-cycler (Regksh-E1teg Ce! ik, Yogteak, ST)]. The PCR product was gel-purified using the rTAcSk ™ PCR product purification kit (^ ASEN Οι; · ιΙκ \\ όγ11ε CA), according to the manufacturer's instructions. The purified PCR product was digested with NbeI and NshbSh and then gel purified using a ΩΙAztec ™ gel extraction kit (ОΙАССЕ, Οι; · ιΙκ \\ όγ11ε CA) according to the manufacturer's instructions. The PCR product isolated from the gel was ligated into pAMC11 (AO 95/26746) and they transformed E. coli EM15 cells (ATCC 55765).

2. κΤΝΡΚ-Ι 2.6B / S105.

PCR-Ι 2.6B / C105 PCR amplification was performed using κΤΝΡΚ-Ι 2.6B / C106 plasmid DNA as a template and the following PCR primers:

OE1SO # 3: (ZEO GO ΝΟ: 70)

5’-ASTSSA ООАТССОСОААААТААТААСОАТССООТСС -3 ’ОЫОО # 4: (ЗЕО ГО ΝΟ: 71)’ -СОООССОСАТССТАТСААААААСОССОААААА & ТТТТС-З '

OYUO # 3 and OYSO # 4 contained the sites of the restriction of WhAT and mutation Ν (105) after the stop codon. The oligonucleotides were designed to overlap the entire matrix to create a new WatCh restriction site for ligation. PCR amplification was performed for 35 cycles; 10 cycles, each of which consisted of 10 s at 92 ° С for denaturation, 30 s at 55 ° С for annealing, and 4 min at 68 ° С for elongation, followed by 25 cycles, each of which consisted of 10 s at 92 ° C for denaturation, 30 s at 55 ° C for annealing, and 4 min + 20 s for elongation [model 2400 thermal cycler (Regksh-E1teg ΟοΙιΐδ, No. r \ ua1k, ST)]. The PCR product was gel purified using an O1Ac gel extraction kit. | Shsk ' ^{| L |} (О1АСЕ№ СНа ^ уОПН, СА) according to the manufacturer's instructions, restrict with WatH1, extract with phenol / chloroform and precipitate with ethanol. Then it was resuspended, ligated into pAMO11 and transformed with them E.coH EM15 cells.

3. δΤΝΕΒ-Ι 2.6Ό / Ν105.

PCR amplification δΤΝΕΒ-Ι 2.6Ό / Ν105 was performed using plasmid DNA δΤΝΕΒ-Ι 2.6E / C106 and the following PCR primers as a template: 5 ’NOOOO # 5: (5E <E GO ΝΟ: 72)

5’-SCHOTTASSSSATATSOASASS & TTTSSSSSSSSAA-3 ’

5 ’SENSE # 6: (5E <E Ιϋ ΝΟ: 73)’ -SSSSSATSSSTATTAATTOAASSASSOAAAASS-3 ’

OYUO # 5 and OYUO # 6 contained the restriction sites NbeI and VatSh and hybridized with the 5 'and 3' ends of the truncated gene, respectively. PCR amplification was performed for 30 cycles; each cycle consisted of 45 s at 95 ° C for denaturation, 1 min at 65 ° C for annealing, and 2 min at 72 ° C for elongation [model 2400 thermo-cycler (Regkt-E1teg Ce! u8, No. r \ ua1k, ST)] . PCR products were purified using a DNA purification system A | / agb ' ^{| L |} (Rgoteda, MabEop, ΑΙ), according to the manufacturer's instructions. The purified PCR products were digested with NbeI and WatSH, extracted with phenol / chloroform and precipitated with ethanol. Then they were resuspended, ligated into pAMO11 and transformed with them E.coH EM15 cells.

Based on the above description of the present invention, a skilled person will understand that various materials and methods can be used or adapted for convenient expression in host cells (eg, E. coH and other bacteria).

4. δΤΝΕΒ-Ι 2.3Ц / b18: δΤΝΕΒ-Ι 2.3Ц / b8 and δΤΝΕΒ-Ι 2.3ϋ / ά15.

PCR amplification δΤΝΕΒ-Ι 2.3C / b18; δΤΝΕΒ-Ι 2.3C / b8 and δΤΝΕΒ-Ι 2.3C / b15 were performed using δΤΝΕΒ-Ι 2.6E / C 106 and the following PCR primers as the plasmid DNA template.

PCR primers for δΤΝΕΒ-Ι 2.3C / b8:

5 ’OYOO # 7: (8Εζ) GO ΝΟ: 74)

USSSATATOTATATSSASSSSSTAAAAAAT-3 ’

5 ’SENSE # 8: (5E <E GO ΝΟ: 75)’ —SSCAASSTPTASAOASASSAATTSAASSASTO-Z’

PCR primers for δΤΝΕΒ-Ι 2.3C / b15:

5 ’О1УО # 9: (5Е <Э ГО ΝΟ: 76) 5’-ССССАТАТОСОСА1ТАОСТСТАССААТСССАСАААС & -3’ 5 ’О1ЛОО # 10: (5Е <2 ГО ΝΟ: 77) 5'-СССААОСТТТТАСААА6ААТТААААСОС

PCR primers for δΤΝΕΒ-Ι 2.3C / b18: 5 'O1LOO # 11: (8Е <Э Го ΝΟ: 78) 5'-ССССАТАТАТОТАССАСССАААСОА-3' 5 'ОСОО # 12: (8ЕО GO ΝΟ: 79) 5'-СССААААСОТАТАТАТАСТАСАТАТАТАТСОСА -3 '

Each of the oligonucleotides OHYO # 7, OHYO # 9 and OHYO # 11 contained the Cbe restriction site, and the oligonucleotides OHYO # 8, OHYO # 10 and OHYO # 12 contained the restriction site Hsh. PCR amplification was performed for 25 cycles; each cycle consisted of 45 s at 95 ° C for denaturation, 1 min at 65 ° C for annealing, and 2 min at 72 ° C for elongation [model 2400 thermo-cycler (Regksh-E1teg Ce! ik, No. r \ ua1k, ST)] . PCR products were purified using a Αί / agb '™ DNA purification system (Rgoteda, Mab1cop, ΑΙ) according to the manufacturer's instructions. The purified PCR products were restricted with NbI and HbCl, extracted with phenol / chloroform and precipitated with ethanol. Then, they were resuspended, ligated into pAMO11, and E.coH EM15 cells were transformed with them.

B. Preparation of products in E. col cells.

Initially, a small freshly cultured inoculum of the desired recombinant clone E.coN carrying the construct for expression of δΤΝΕΒ-Ι 2.6Ό / Ν105, δΤΝΕΒ-Ι 2.6C / C105, δΤΝΕΒΙ 2.6C / C106, δΤΝΕΒ-Ι 2.3ϋ / ά18, δΤΝΕΒ-Ι 2.3C / b8 and δΤΝΕΒ-Ι 2.3Ό / 615, were obtained by transferring the entire contents of the freezing ampoule (1.5 ml in volume) to a 2 L flask containing 500 ml of Luria broth. The culture was incubated on a rotating shaker at 37 ° C and 350 rpm. The density of the culture was determined by measuring the absorbance at 660 nm (OI ₆₆₀ ). A seed culture was grown to a density of> 2.0 OI ₆₆₀ when 125 ml of the culture was aseptically transferred to a 15 liter industrial fermenter containing 10 L of sterile growth medium.

The batch medium and the fermentation conditions used for industrial fermentation are described by ΞπίίΤ (1993) in the work of SNet1s11uEeGteb Mebst Toh She STegrgobisNop Ot e SesotYpap! Rgo! Yesht E. Soy, Abstracts of Colorado State University. In this work, the use of a complex medium containing casein hydrolyzate, salt, glycerin and antifoam, which is sterilized in a fermenter, is recommended. After the fermenter has cooled to a temperature below 40 ° C, microelements and thiamine hydrochloride are sterilized by filtration.

When the medium temperature stabilized at 37 ° C, a seed culture was inoculated into the medium. The growth of the culture was monitored, determining OI ₆₆₀ . The culture was maintained at pH 6.0 by the automatic addition of 5M sodium hydroxide and 5M hydrochloric acid. When the ΟΌ ₆₆₀ value was in the range of 9.510.5, the culture was induced by aseptic addition of sterile isopropyl β-Ό-thiogalactopyranoside (ΙΓΤΟ) to a final concentration of 0.50 mM. The culture was harvested at the end of growth.

The culture medium and growth conditions corresponded to those described by 8n1GG (1993), Kirg, with the following exceptions: ammonium sulfate (2.0 g / l) and L-cysteine hydrochloride monohydrate (1.0 g / l) were added to the medium; tetracycline hydrochloride was absent in the medium; The pH was maintained at 6.0 by the addition of sodium hydroxide and hydrochloric acid; growth temperature increased to 37 ° C; the inductor concentration was increased from 0.15 to 0.50 mM ΙΓΤΟ and the time of culture collection was determined based on growth retardation, and not on the time elapsed since the induction.

Upon completion of the fermentation, cells were collected by centrifugation in 500 ml vials. Cells were pelleted by centrifugation at 10,000 rpm for 30 minutes. The resulting cell paste was diluted to 15% solids in destructive buffer, consisting of 50 mm Τπδ and 5 mm EDTA with pH 8.0. Suspended cells were lysed by passing the solution three times through a homogenizer (AUR 6a1pcs Shs., Eyegey, MA) with a pressure of 55158080 kPa. The homogenate was centrifuged at 10,000 rpm for 30 minutes to isolate teleinclusions. The inclusion bodies were washed by resuspending in destructive buffer and centrifuging the solution a third time at 10,000 rpm for 30 minutes. The inclusion bodies were resuspended in deionized water (1: 1 ratio) and centrifuged for the last time at 10,000 rpm for 30 minutes for a second wash. Dedicated, washed Taurus inclusions were ready for solubilization, re-laying and cleaning. Each selection yielded approximately 200-250 g of teleinclusions.

In an alternative embodiment, fermentation to produce truncated κΤΝΡΒ-Ι can be carried out as follows.

An initially small, freshly cultured inoculum of the desired recombinant E. cloy clone carrying the construct for expressing κΤΝΕΚ-Ι 2.6Ό / Ν105 or κΤΝΡΚ-Ι 2.6P / C106 was obtained by transferring the entire contents of the freezing ampoule (1.5 ml) to 2-L flask containing 500 ml BBB of yeast extract (10 g / l), pH 7.0. The culture was incubated on a rotating shaker at 33 ° C and 300 rpm. The density of the culture was determined by measuring the absorbance at 660 nm (ΟΌ ₆₆₀ ). The seed culture was grown to a density> 2.0 ΟΌ ₆₆₀ , when 80 ml of the culture was aseptically transferred to a 15 liter industrial fermenter containing 7 L of sterile growth medium.

For industrial fermentation, a feed batch process was used. Batchfermentation medium is a complex medium containing yeast extract, salts and antifoam that is sterilized in a fermenter. After the fermenter has cooled to a temperature below 40 ° C, microelements sterilized by filtration, glucose, magnesium sulfate and hexametaphosphate are added. Two top dressings were used, the first containing carbon (glucose / magnesium sulfate) and the second containing nitrogen, the source of which was yeast extract.

When the temperature of the batch medium stabilized at 33 ° C, an inoculum of the inoculum culture was introduced into it. Culture growth was monitored, determining ΟΌ ₆₆₀ . The pH of the culture was maintained at 7.0 by the automatic addition of ammonium hydroxide and 48.7% citric acid. When the value of ΟΌ ₆₆₀ was in the range of 8.0–12.0, feeding Ι was started with an exponentially increasing feed rate. When the value of ΟΌ ₆₆₀ was in the range of 30–40, feeding ΙΙ was started at a constant feed rate. When the ΟΌ ₆₆₀ value reached 67–83, the culture was induced by aseptic addition of a sterile autoinducer (homoserinlactone) to a final concentration of 0.6 mg / L. At the induction stage, the feed rates Ι and top dressings ΙΙ were made constant. The culture was harvested 16 ± 2 hours after induction.

Upon completion of the fermentation, cells were collected by centrifugation in 500 ml vials. Cells were pelleted by centrifugation at 10,000 rpm for 30 minutes. The resulting cell paste was diluted to 15% solids in destructive buffer consisting of 50 mM Τπκ and 5 mM EDTA with pH 8.0. Suspended cells were lysed by passing the solution three times through a homogenizer (AUR 6Ai1Sh Shs., Eyegey, MA) with a pressure of 55158080 kPa. To isolate Taurus inclusions, the homogenate was centrifuged at 10,000 rpm for 30 minutes. The inclusion bodies were washed, resuspended in destructive buffer, and the solution was centrifuged a third time at 10,000 rpm for 30 minutes. The inclusion bodies were resuspended in deionized water (1: 1 ratio) and centrifuged for the last time at 10,000 rpm for 30 minutes for a second wash. Dedicated, washed Taurus inclusions were ready for solubilization, re-laying and cleaning.

C. Solubilization / re-laying.

The washed inclusion bodies obtained by 10-liter fermentation were solubilized in 800 ml of solubilization buffer (50 mM Τγϊκ, 8 M urea, 160 mm cysteine, pH

9.5). The pH of the solubilization mixture was adjusted to pH 9.5 by addition of 10Ν Ν; · ιϋΗ and the mixture was stirred at room temperature for 2-3 hours. Approximately 200-250 g of Taurus inclusions were isolated at each formulation.

Each solubilization mixture was diluted 1:20 with cold renaturation buffer (50 mM ίτίκ, 1.1 M urea). The final volume was about 16 liters. Then the pH value of each mixture was adjusted to pH 9.7 by the addition of 6Ν HCl, and the mixture was slowly stirred at 4 ° C for 2-3 days.

Then, the pH of each mixture was adjusted to pH 5.0 by the addition of glacial acetic acid and 6Ν HCl. A precipitate formed in each mixture, which was removed by centrifugation at 10,000 d using a Vesktap 12H8 centrifuge. Then, each sample was filtered through 5 μm and 0.22 μm filters.

Ό. Cleaning up.

The material obtained as a result of re-laying was ready for cleaning on a ΙΧ-1 8P-8erBagoke V1d Beab ™ column (RBattaaa B1o1esN, Shs., P1ksa1a ^ ay, Ν1).

Column 1-1 8P-8er11agoke V1d Weab ™ (4.4 cm x 20 cm).

Buffer a Buffer b 25 mm acetate 25 mm acetate 50 mM NaCl 375 mM NaC1 pH 5.0 pH 5.0

The column was balanced with buffer A in a volume equal to 4-5 column volumes, before separately applying each sample of material after re-laying. Each material sample was applied to the column separately. At each application, no more than twelve grams of protein per liter of resin was applied to the column. At each application, the column was washed with 3-4 volumes of buffer A (until the UV absorbance returned to its original value). At each application, the protein was eluted from the column with a linear gradient (8 column volumes) of increasing concentration from 50 to 375 mM NaCl. The entire protein peak from each application was pooled. Each protein peak began to be harvested when UV absorption reached 20% of the maximum value. The collection was stopped when the absorption reached 50% of the maximum value or the absorption ceased to decline.

Current speed

- 7.5 post rpm for balancing and washing,

- 15 post. vol./h for application,

- 6 post. rpm for elution.

All stages of column cleaning were carried out at 4 ° C.

Each ΙΧ-1 pool was ready for cleaning on a Shuo Reag1 ™ Vi! U1 650M GC column (% Naak, PF1abe1rFa, RA).

300 ml Shuo Reag1 ™ Vi! U1 650M AL column (4.4 cm x 20 cm).

Buffer a Dilution buffer Buffer b 20 mM \ aP () | 40 mM ^ PO ₄ М1Ш (,) Н ₂ О 1.8 M \ aCl pH 6.0 4M \ aC1 pH 6.0

The column was balanced with buffer A in a volume equal to 4-5 column volumes before separate application of each ΙΧ-1 pool. Each ΙΧ-1 pool was diluted 1: 1 with dilution buffer and adjusted to pH 6.0. For each application, one diluted ΙΧ-1 pool was applied to the column. At each application, no more than ten grams of protein per liter of resin was applied to the column. At each application, the column was washed with three volumes of buffer. Protein was eluted from the column by a linear gradient (8 column volumes) of decreasing concentration from 1.8 M # 1C1 to H ₂ O. Each protein peak was started to be collected when UV absorption reached 15-20% of the maximum value. The collection was stopped when the absorption reached 50% of the maximum value or the absorption ceased to decline.

Current speed - 6 post. vol./h for balancing, applying and washing, 3 post. rpm for elution.

Each AL pool was ready for concentration / diafiltration.

Concentration / diafiltration (C / Ό).

For each C / ϋ stage of each GC pool, 1 square foot of regenerated PBC ™ cellulose membrane with cutoff MV 5000 (M1Sh-Roge, WebGogb, MA) was used. Each GC pool was concentrated to a volume of about 200 ml and then diafiltered against 6-7 volumes of 20 mM LaPO ₄ , pH 6.0, until the conductivity reached values <4 mm per hour.

Each concentration / diafiltration step was carried out at room temperature.

Each C / ул pool was ready for cleaning on the ΙΧ-2 - 365 8P-8erNagoke HP ™ column (RNagtas1a B | o1es11, Sh., P1ksa1a ^ ay, Ν1).

Column ΙΧ-2 - 365 8P-Sepharose HP ™ (5 cm x 18.5 cm).

Balancing buffer Buffer a Buffer b 20 mM ^ PO ₄ 20 mM ^ PO ₄ 20 mM HaRo ^ pH 6.0 pH 6.3 50 mM aC1 pH 6.8

The column was balanced with equilibration buffer A in a volume equal to 4 column volumes before dividing each C / ϋ pool. Each C / ϋ pool was applied to the column at the rate of not more than eight grams of protein per liter of resin. At each application, the protein was eluted from the column by a linear gradient (8 column volumes), pH from 6.3 to 6.8, and 1M - 0M NaCl salt (buffer B). The collection of fractions began at 1.0 ΟΌ at the beginning of the peak and ended at a value equal to 50% of the maximum.

In an alternative embodiment of the invention, the truncated kT # K-1 may be solubilized, repackaged, and purified as follows.

C. 1. Solubilization / re-laying.

The washed inclusion bodies were solubilized with 8 M urea, 60 mM Tpc, 100 mM cysteine to obtain a final concentration of 6.5 M urea, 50 mM Tpc and 80 mM cysteine, pH 9.4 and 5-10 mg / ml truncated kT№K- one. The material was stirred at room temperature for 90 min, and then subjected to re-laying, diluting 1:10 with a cold (4-8 ° C) solution of 0.85 M urea, 50 mM TPC pH 9.8 (pH measurements were carried out at 4-8 ° C) .

The solution for re-laying was stirred for 24-72 hours at 4-8 ° C. At the end of this period, glacial acetic acid (~ 20 mM) was added and the pH was adjusted to 5.0. The resulting precipitate was removed by centrifugation, and the supernatant was stored for application to the first column.

Ό.1, Cleaning.

After acid precipitation, the clarified pool was applied onto an 8P-Sepharose B1d Weab ™ column (Rbattasha Vu! Esb, 1 ps., P1xa! A ^ ay, ΝΡ), balanced with 20 mM sodium acetate, 75 mM No. C1, pH 5.0. No more than 15 g of truncated kT # K per liter of resin was applied to the column. After application, the column was washed with 20 mM sodium acetate, 75 mM No. C1, pH 5.0, in a volume equal to 3 column volumes, and was eluted with a linear gradient (9 column volumes) from 75 mM to 450 mM No. 1C1 in 20 mM acetate, pH 5.0. The entire stage on the column 8P-Sepharose V1d Weab ™ (8P-BB) was carried out at 4-8 ° C.

The 8P-BB pool was diluted in the ratio 1: 1 2M No. C1, 60 mM acetate, pH 4.5, and, if necessary, the pH was adjusted to 4.5. The diluted 8P-BB pool was loaded onto a Touoreag1 ™ Vi1u 1 650M column (Toko Naak, Pb11abe1rb1a, RA), which was balanced with 1M No. C1, 60 mM acetate, pH 4.5. 10–13 g of truncated KΤNΕΚ-I per liter of resin was applied to the column. After application, the column was washed (3 column volumes) with 1M No. C1, 30 mM acetate, pH 4.5 and eluted with a linear gradient (8 column volumes) with 1M 0M No. 1C1 in 30 mm acetate, pH 4.5.

The fractions of purified truncated KΤNΕΚ-I from a Vi1u 1,650M column were combined, diluted 1: 5 with water and applied to an 8P-Sepharose Н1db RegGogtapse ™ (8Р-НР) column (Rbagtasha Vu! Esb, 1 ps., P1ksa! A ^ ay, ΝΡ) , balanced with 30 mm acetate, pH 4.5 (load no more than 15 g / l of resin). Then the column was washed with 30 mm sodium acetate, pH 4.5, in a volume equal to 3 column volumes, and was eluted with a linear gradient (12 column volumes) from 100 to 400 mm No. 1 C1 in 30 mm acetate, pH 4.5. The fractions of purified truncated kΤNΕΚ-I were combined and the pH was adjusted to 5.0 by the addition of NO.

C. pegylation.

1. Obtaining kT№K-1 2.6O / M05 -! - VTsRES (33 kD).

To a cooled (4 ° C) stirred solution of kT№K-2.6O / M05 (3.5 mg / ml) in 50 mM sodium acetate, pH 4, a 3-fold molar excess of! -ViREO (mono-! - butoxypolyethylene glycol, average molecular weight = 33 kD, 8 beaoeg! er Po1utegk, 1 ps.). Then, NaCNΒΗ3 was added to a final concentration of 20 mM, and the reaction mixture was stirred at 7 ° C for 18-24 h.

The degree of protein modification during the reaction was monitored by 8ES НРЬС using a Т8КС3000 _8УХЬ column (Toko Naak, Mop! Reached She, RA), eluting with 0.1 M sodium phosphate buffer, pH 6.9, 0.5 M No. 1Cl and 10% ethanol at a rate of 0.7 ml / min (Toko Naak, Mop! dote ^ 111e, RA).

The pH of the reaction mixture was adjusted to 3.5 1M HC1, and then the reaction mixture was diluted with water to a final protein concentration of 1.5 mg / ml. 8Т№К-1 2.6О / М05 -! - ВЦРЕС (33 кОа) was separated from the excess! -VIREO and other reaction by-products using an 8P Sepharose НР 16/10 ™ ion-exchange chromatographic column (Pbtas1a Vyu! Esb, 1 ps., P1xa ! a ^ ay, Νί). The reaction mixture was applied to a column and unreacted! -ViREO was eluted with three column volumes of initial buffer A (20 mM sodium acetate, pH 4.0). 8Т№К-1 2.6Ό / Ν105-! VTsREO (33 KOa) was eluted (20 volumes) with a linear gradient of 0-30% buffer B (1M No. 1С1 in 20 mM acetate, pH 4.0). The absorption of the eluate was controlled at 280 nm. Each fraction containing kT№K-1 2.6О / М05 -! - ВЦРЕС (33 KOa) was analyzed with 8E8-PACE using ready-made 4-20% gradient gels (Nονеx, 8ап О1едо, СА). Based on the results of analysis of 808-PACE, the fractions were combined, concentrated and sterilized by filtration. Each final pool of purified kT№K-1 2.6Ω / Ν 105-1-ViRES (33 KOa) was again analyzed by 8O8-PAOE and 8ES-NRS. This protein was dissolved in 10 mM sodium phosphate, pH 6.5 and 20 mM N801.

2. Obtaining kT№K-1 2.6Ώ / Ν105-33 kD (MeRES).

To a chilled (7 ° С) stirred solution of kT№K-1 2.6Ώ / Ν105 (4 mg / ml) was added 10% acetic acid until a pH of 5.0 was reached. To this solution were added 15 mM NaCNΒΗ3 and a twofold molar excess of! Butoxy PEC (! -Butoxypolyethylene glycol, average molecular weight = 33 kD, 8 beag! Po1uteg, 1 ps.). The reaction mixture was briefly stirred and then incubated at the same temperature for ~ 18 hours.

Ί5

Ί6

After 18 hours of incubation, the pH in the reaction mixture was adjusted to 3, by adding citric acid.

δΤΝΡΚ-Ι 2.6Ό / Ν1ο5-Μ € ΡΒ6 (33 kDa) was separated from the excess MePEC and other reaction by-products by ion exchange chromatography on an 8P-Sepharose HP ™ column (Ryagtas1a Βίοίοοίι, Sch., ΡίδοαΡηναν, Ν).

The reaction mixture was applied to a column (not more than 8 mg / ml resin) and unreacted MeEPEC was eluted with three column volumes of start buffer A (2ο mM sodium citrate, pH 3, ο). δΤΝΡΚ-Ι 2.6 ШХ 1 (Е-МеРЕС (33 kDa) was eluted with a linear gradient (16 column volumes) ο, 1 - ο, 5 M NaCl in 2ο mM citrate, pH 3, ο. The eluate was monitored at 28ο nm. Each fraction containing δΤΝΡΚ-Ι 2.6Ό / Ν1ο5MePBO (33 kOa), 8E8-PACE was prepared using ready-made 4-2% gradient gels (Yueh, 8ap O1edo, CA). Based on the results of the 808-PACE analysis, the fractions were combined, concentrated and sterilized Each final pool of purified δΤΝΡΚ-Ι 2.6Ό / Ν1ο5-MEPEC (33 kD) was again analyzed with 808-PACE. The purified δΤΝΡΚ-Ι 2.6Ω / Ν 1 () 5-MePEC (33 kD) was concentrated to 5-2ο mg / ml and was dissolved either in PB8, pH 6.5 (1ο mM sodium phosphate, 35-1οο mM NaCl), or in 2ο mm acetate, 1ο mM NaCl, pH 5, ο.

3. Preparation of δΤΝΡΚ-Ι 2.6О / Х1 () 5-МеРЕС (2ο кД).

The procedure of step A to obtain δΤΝΡΚ-Ι 2.6О / Х1 () 5-МеРЕС (33 kD) essentially repeated the above, with the only difference being МеРЕС (monomethoxypolyethylene glycol, average molecular weight = 2ο KD, 811 eag \\ 'a1eg Po1utega, Sch.) Was replaced by MeEPES (monomethoxypolyethylene glycol, average molecular weight = 33 kD, 811eag \\ 'a1eg Po1utega, Sch.). This protein was dissolved in 1 mM sodium phosphate, pH 6.5, and 2 mM NaCl.

Obtaining additional conjugates.

Additional conjugates δΤΝΡΚ-Ι 2.6О / Х1 () 5-МеРЕС were obtained in almost the same way as δΤΝΡΚ-Ι 2.6 ^ / N105-МеРΒС (33 kD), with the difference that the following types of PEC aldehydes were used (811еag \\ 'a1eg Ro1utega, Sh.):

monofunctional linear - MV 5 kD, 6 kD and 57 kD;

branched monofunctional - MV 1ο KD, 2ο KD and 4ο KD;

linear differential - MV 8 kD and 2ο kD;

branched trifunctional - MV

Yu cd.

These proteins were dissolved in 1 mM sodium phosphate, pH 6.5, and 2 mM NaCl.

5. An alternative method of pegylation.

In an alternative embodiment, truncated δΤΝΡΚ-молекулы molecules can be pegylated and purified as follows.

The 8P-HP eluate (3-5 mg / ml, pH adjusted to 5, ο) was reacted with two moles of polyethylene glycol (for example, MePEC or 1-ViPEC) per mol δΤΝΡΚ-Ι 2.6Ο / Ν1 () 5 (~ 5 g 1-ViRES per gram δΤΝΡΚ-Ι 2.6Ο / Ν1 () 5). After the polyethylene glycol was dissolved, 1ο-2οmM sodium cyanoborohydride was added and the solution was incubated overnight at 7-15 ° C. At the end of the RESING reaction (~ 18 h), the reaction was stopped by adding 1ο mM glycine. The PEGing mixture was diluted with four volumes of 5ο mM acetate, pH 4.ο, if necessary, adjusted to pH 4, ο and applied to an 8P-HP column equilibrated with 5ο mM acetate, pH 4, ο. No more than ~ 8 g of δΤΝΡΚ-Ι 2.6Ώ / Ν1ο5 per liter of resin was applied to the column. After application, the column was washed (3 column volumes) with equilibration buffer, pH 4.5, and eluted with a linear gradient of 0M - ο, 3 M NaCl in 5ο mM acetate, pH 4, ο. Fractions of monopaginated δΤΝΡΚ-Ι 2.6Ώ / Ν1ο5-3ο кОа were collected, adjusted to pH 5, ο, concentrated and diafiltered into the final isotonic buffer. All purification steps were carried out at room temperature. This protein was dissolved either in PB8, pH 6.5 (1ο mM sodium phosphate, 35-1οο mM No. 1C1), or in 2ο mM acetate, 1οο mM NaCl, pH 5, ο.

6. Obtaining dimerized (b) δΤΝΡΚ-Ι 2.6I / СУ5 and dimerized δΤΝΡΚ-Ι 2.6О / С1 () 6.

Sulfone-Activated Polyethylene Glycol (Obtained and Purified in accordance with US Patent Application No. ο8 / 473,8ο9 filed June 7, 1995 and US Patent Application No. ο8 / 611,918 filed March 6, 1996) | RES 20,000 bis-vinylsulfone], was used for dimerization of proteins in accordance with the method described in published application XV Ο 95/34326, except for the recovery procedure and reaction conditions. Before attaching polyethylene glycol, the proteins were reduced by adding 4 mol мол per 1 mol of protein at 5-6 ° C, pH 7.6. All reactions were carried out in the presence of 3% glycerol. The dimerized proteins were designated δΤΝΡΚ-Ι 2.6I / СУ5йЬ and δΤΝΡΚ-Ι 2.6О / С1 () 6йЬ. These proteins were dissolved either in PB8, pH 6.5 (1ο mM sodium phosphate, 35-1οο mM No. 1С1), or in 2ο mM acetate, 1οο mM NaCl, pH 5, ο.

7. Obtaining comparative molecules δΤΝΡΚ-Ι.

(ί) δΤΝΡΚ-Ι 4Ω / Ν1ο5 was obtained as described in EP 422339. 8ΤΝΡΚ-Ι 4 ^ / N105-ΐ-ViPΒC (33 kD) was obtained by RESING δΤΝΡΚ-Ι 4Ω / Ν1ο5 in accordance with the above procedure for pegylation δΤΝΡΚ-Ι 2.6 ^ / N105-В-ViRΒS (33 kD). 8ΤΝΡΚ-Ι 4Ι2 / Ν1 (EX-OMeRES (33 kD) was obtained by pegylation δΤΝΡΚ-Ι 4Ο / ΝΚΙ5 in accordance with the above procedure for pegylation δΤΝΡΚ-Ι 2.6Ό / Ν 105 -! - МеРЕС (33 kD). 8ΤΝΡΚ-Ι 4I / C105 and δΤΝΡΚ-Ι 4P / C105bb were prepared as described in published application AO 95/34326. This protein was dissolved in 10 mM sodium phosphate, pH 6.5 and 20 mM No. 1C1.

(ίί) δΤΝΡΚ-Ι 4I / C105-33 kD (MePEC) was obtained by pegylation with 4I / C105 in accordance with the above procedure for pegylation δΤΝΡΚ-Ι 2.6I / C105-33 kD (MePEC) with the difference that the reaction was carried out at pH 7 , 5 with 1.3 moles ΌΤΤ per mole of δΤΝΡΚ-Ι for 5-6 hours, followed by removal of ΌΤΤ on the 8P-Sepharose ™ PP column and pegylation with 1.5-3 moles of PECA per mole of protein for at least 15 hours at room temperature. This protein was dissolved either in PB8, pH 6.5 (10 mM sodium phosphate, 35-100 mM No. 1C1), or in 20 mM acetate, 100 mM NaCl, pH 5.0.

(ΐϊϊ) δΤΝΡΚ-Ι 3Ό / Ν105 (truncated by 34 amino acids at the C-terminus of δΤΝΡΚ-Ι 4Ό / Ν105) was prepared as follows. PCR amplification was performed using δΤΝΡΚ-Ι 4Ό / Ν105 and OYUO # 13 and OYUO # 14, which encode the restriction sites Νώ; Ι and НшбШ, respectively, and hybridize with the 5 'and 3' ends of the truncated gene, respectively. PCR amplification was performed for 25 cycles; each cycle consisted of 30 s at 94 ° C for denaturation, 15 s at 60 ° C for annealing, and 1 min at 72 ° C for elongation [model 2400 thermo-cycler (Regkt-E1seg Ce1i5, Nogo1k, ST)]. The PCR product was purified using a PCR product purification kit P ^ cssk ™ OACEN C11a15 \\ 'og111, cA).

The PCR product isolated from the gel was ligated into pAMC11 and transformed with E. coli PM15 cells.

5 ’HYSISTIC # 13: (8E <E GO ΝΟ: 80)

5'-OOTTAAOSSATATOOASASSOTTTOSSSSSSAA-3 '

3 'OYOO # 14: (5Εζ> GO ΝΟ: 81) 5’-СССААОСТТТТАОООСОСАССОСОТТТТТТТТТ-3'

This protein was dissolved in 10 mm sodium phosphate, pH 6.5, and 20 mm No. 1C1.

(ίν) δΤΝΡΚ-Ι 3I / C105 (truncated by 34 amino acids at the C-terminus of δΤΝΡΚ-Ι 4I / C105) was obtained in much the same way as 8ΤΝΡΚΙ3Ό / Ν105, with the difference that δΤΝΡΚ-Ι 4Ό005.8ΤΝΡΚ- served as a matrix Ι3Ό / α05 was dissolved either in PB8, pH 6.5 (10 mM sodium phosphate, 35-100 mM No. 1C1), or in 20 mM acetate, 100 mM NaCl, pH 5.0.

(ν) δΤΝΡΚ-Ι 3I / С105bЬ was obtained almost in the same way as 5ΤΝΕΕ-Ι4Ό / Ο056Κ with the difference that δΤΝΡΚ-Ι 3I / С105 was used as the starting material instead of δΤΝΡΚ-Ι 4I / С105. δΤΝΡΚ-Ι 3I / C105bb was dissolved either in PB8, pH 6.5 (10 mM sodium phosphate, 35-100 mM No. 1C1), or in 20 mM acetate, 100 mM NaCl, pH 5.0.

Example ΙΙ.

Various forms of truncated, recombinant soluble ΤΝΡΚ-Ι were tested for their ability to inhibit TIR activity.

A. Cytotoxic test on AES cells.

The test on AES cells is a test for the proliferation of cells ν ίΙΐΌίΙΐΌ (Eb ^ agbk e! A1., (1991) Ebosgto1ou. 128: 989-996). This cell line is sensitive to ΤΝΡ-α (i.e., ΤΝΡα is cytotoxic). In the presence of an ΤΝΡ-α inhibitor, cells are protected from the cytotoxic effect and are able to proliferate.

Protocol.

ΤΝΡ-sensitive cells of AESH 164 clone 13 (ATCC, EosktShe, MI) were suspended at a concentration of 20x10 ⁴ cells / ml in ΚPMI medium (C1bco, Sagib Schapb, ΝΥ) with the addition of 5% fetal calf serum (Nus1ope, Odbep, ϋΤ) and penicillin 50 U / ml: streptomycin 50 mg / ml. One hundred microliters of the cell suspension was added to each well of 96-well flat-bottomed microtiter plates and the cells were allowed to adhere for 4-6 hours at 37 ° C in an atmosphere of 5% CO ₂ . 10 μl of 0.0060 mg / ml actinomycin-Ό (8ft Sjeslca1 Co., 8! Bosh5, MO) was added to each well. Then, 10 μl of recombinant human α-α at a concentration of 50 ng / ml (final concentration of 5 ng / ml) was added to each well. After the recombinant human α-α was added to the wells, (10 μl / well) serial two-fold dilutions of various δΤΝΡΚ forms prepared on PB8 (δΤΝΡΚ-Ι 2.6I / C106, δΤΝΡΚ-Ι 41) / C10 and δΙ-Ι 4I / C105b) were added to the wells . AES cells 164 of clone 13 were incubated for 18 h at 37 ° C in an atmosphere of 5% CO2. After incubation, 10 μl (2 mg / ml) of an MTT tetrazolium organic dye solution (3- [4,5-dimethylthiosol-2-yl] -2,5-diphenyl tetrazolium bromide; 8% Seschlossa Co.,

8! Bosh5, MO) and the cells were incubated for another 4-6 hours. The cells were solubilized by adding 50 μl of solution 1) \ 11 ^; / 81) 8 (20% 8Ό8 and 50% Ν, Ν dimethylformamide, pH 4.7). The PMR / δΌδ solution was pipetted several times until the MTT crystals were completely dissolved and the cells were incubated for another 2-22 hours. Absorbance values (ab) were determined on a U _shah reader at a wavelength of 570 nm. The percentage of specific cytotoxicity was calculated on the basis of the optical density according to the formula:% specific cytotoxicity = [abk (cells + medium) - abk (cells + sample)] / [abk (cells + medium) - ab (cells + TX-100)] . The number of units ΤΝΡ in each sample was determined using the percentage of specific cytotoxicity of the mouse standard, as described previously.

The results of the cytotoxic test on AES cells are shown below in table. 2.

table 2

The activity of 1p Uygo in the test on cells No. ESh

Compound ΙΟ ₅₀ , ng / ml δΤΝΕΚ-1 2.6B / S106 208 δΤΝΕΚ-1 4B / C105 238 δΤΝΕΚ-1 4B / C105bb Ν / A

Judging by the results of the cytotoxic test on νΞΗΙ cells, no significant differences were found between 8ΤΝΕΚ-1 2.6E / C 106 and 8ΤΝΕΚ-1 4E / C105 with respect to the bioefficiency of 1n yugo.

B. Cytotoxic test on b929 cells.

The cytotoxic test on L929 cells is a test for the proliferation of hell cells (Ragteu e! A1., (1993), I. Itipo1., 151: 389-396), which also allows one to evaluate β-dependent cytotoxicity. Cell lines are sensitive to ΤΝΕ-α (i.e., ΤΝΕ-α is cytotoxic). In the presence of a soluble ΤΝΕ-α inhibitor, cells are protected against cytotoxicity and are able to proliferate.

Protocol.

The b929 cell line was obtained from the American Collection of Cell Cultures (catalog number Cb 1, NCC clone 929, clone strain L, connective tissue, mouse). To maintain cells, we used KPMI 1640 medium supplemented with 10% fetal calf serum, 1% L-glutamine solution, and 1% penicillin-streptomycin solution.

In the experiment, 96-well microtiter plates (Sogspd) were used, and only 60 internal wells were seeded. Each standard and test sample was added to three wells on one plate.

The ΤΝΕ-α used in the experiment was obtained from Κ & Ό 8y8! Et8 (M ^ iiearο1 ^ 8, WE). The final concentration of ΤΝΕ-α used in the experiment was 1 ng / ml in all experimental wells.

The solvent in all experiments was the growth medium for L929 cells, 10 ng / ml S-α and 10 μg / ml actinomycin S (81 dta CHet1ca1 Co., 8! Bosh8, MO).

The material from the dies was collected using an X МЕ / MEN solution (1.5 mg / ml XTT + 75 mM ME ^.

On day 1, cells were seeded in tablets. A cell suspension was prepared by trypsinizing and resuspending the cells to a final concentration of 3.33x10 ⁴ cells / ml. 180 μl of this suspension were seeded in 60 internal wells of the titration plate. 200 μl of growth medium was dispensed into 36 external wells to avoid evaporation-induced artifacts. The plates were allowed to stand at room temperature, covered with foil and left motionless for about 1 hour. The test plates were placed in an atmosphere of 5 ± 1% CO ₂ with high humidity and a temperature of 37 ± 2 ° С. Before serial dilutions of 8ΤΝΕΚ-Ι were added, the plates were incubated for approximately 20-22 hours.

On day 2, prototypes and standard 8ΤΝΕΚ-Ι 4Ό / Ν105 were prepared:

the 8ΤΝΕΚ-Ι 4Ό / Ν105 standard and test samples were diluted to a concentration of approximately 2.0 mg / ml (or other acceptable concentration). Serial dilutions of this concentration were prepared in order to obtain a tenfold dilution curve ranging from 1.0x10 ⁶ ng / ml to 1.0x10 ^-3 ng / ml, including the point 0 ng / ml (solvent only). If other concentrations were acceptable, then these concentrations were used. 1000 μl of each dilution was dispensed into three test wells. After the addition of serial dilutions, the plates were incubated for 20 ± 1 h in an atmosphere of 5 ± 1% CO ₂ with high humidity and a temperature of 37 ° C ± 2 ° C.

On day 3, 50 μl / well of ΧΤΤ / MEK solution was added to 60 internal wells of the experimental plates. The plates were incubated in an atmosphere of 5 ± 1% CO ₂ with high humidity and a temperature of 37 ° C ± 2 ° C in an incubator (Еа1соп, No. \ ν , No. \ ν Wogk) 24 ± 0.5 hours

On day 4, the optical density (ΟΌ) of the experimental plates was determined at a wavelength of 490 nm minus 650 nm using an E8Ascanner (8resAmAH, Veskstap EjitepK Sh., Ei11epop, SA). If at these wavelengths for the wells received values of 4.000 ΟΌ, then the tablet was immediately scanned again at 490 nm minus 650 nm and for the calculations used values of 490 nm minus 650 nm.

The standard logarithmic dose-response curve was constructed using a four-parameter logistic curve. Initial concentrations of unknown samples were calculated from the standard curve, the EE ₅₀ for the standard and the correlation coefficient for the standard curve were calculated.

Results.

The results of the cytotoxic test on b929 cells are shown below in table. 3.

Table 3

The activity of tyugo in the cytotoxic test on b929 cells

Compound Concentration, mg / ml EE50, ng / ml 8ΤΝΕΚ-Ι 4E / S105b 7.8 1.0 ± 0.1 8ΤΝΕΚ-Ι 2.6 1) С105бЬ 2.6 1.1 ± 0.0 8ΤΝΕΚ-Ι 2.6 E / C106b 2.2 1.0 ± 0.1 8ΤΝΕΚ-Ι 4Ώ / Ν105-1- 2.0 229.2 ± 8 ViRES (33 cd) 8ΤΝΕΚ-Ι 2.6E / S105-1- 1.7 210.2 ± 9 ViRES (33 cd) 8ΤΝΕΚ-Ι 4E / S105-1- 1,1 325.5 ± 147 ViRES (33 cd) Internal standard: 3,5 314.8 ± 8ΤΝΕΚ-Ι 4E / S105 188.1

The above results indicate that 8ΤΝΕΚ-Ι 8ΤΝΕΚ-Ι 4E / С105бЬ, 8ΤΝΕΚΙ 8ΤΝΕΚ-Ι 2.61) / 'С‘105с1Ь and 8ΤΝΕΚ-Ι 8ΤΝΕΚ-Ι

2.6E / C106bb are active and they are characterized by similar dose-response parameters when compared with the standard. The data also indicate that kTKHEV-Ι 4V / X105-1-VNRES (33 kD) and kTKHER-Ι 2.60 / C105 -1-ViRES (33 kD) are almost 2 1 code less active, but nevertheless, are still active in this test when compared with CTXER-4I / C105b.

Run # 2 KTINY 3B / S105b 0.2 2.27 ± 0.3 kTKhZhV 3B / S105b 0.2 2.0 * kTKhZhV 3B / S105b 1.9 1.8 * KTINY 3B / Sh05 2,4 413.3 * Internal standard: 3,5 115.9 ± 42.1 KTINY 4B / S105

* Data for one point

The above results indicate that kTDEED 3E / C105bb is active and its values of EI ₅₀ vary within the limits typical for kTDEED 4E / C105bb (run # 1), kTKHEID 2.6I / C105bb (run # 1) and KICHERL 2.6O / C106bb ( run # 1). The data also indicate that kTDEED 3Ό / Χ105 is less active than the internal standard kTDEED 4E / C105.

C. Reactivation model of arthritis induced by streptococcus cell wall.

Tests on the model of arthritis reactivation in rats induced by streptococcal cell wall were performed using the known protocols (Ekkeg e1 a1., (1985), AtShpbk Apb Kieitabkt, 28: 1402-1411 and Makago e1 a1., (1996), Proc. Asab. 8cE I8A, 93: 402-406).

Protocol.

Female Lewis rats weighing 175-185 g (Syat1ec Ktuet Eaboguepek, Sch., Huptcop, MA) were injected intra-articularly into the right ankle joint with a sterile suspension of streptococcus cell wall products containing a peptidoglycan-polysaccharide (8Cxu, bbbbb, bbbbbb 1.5 mg / 10 mg per joint.

As a control, saline was injected into the same joint on the other limb. Intra-articular 8Ci injection causes acute arthritis of relatively short duration with joint swelling reaching a peak one or two days after the injection. After 20 days, during which the inflammatory reaction died away, 8C was again administered by intravenous injection at a dose of 200 mg / 200 ml per rat. The second dose of 8C1 is sufficient for reactivation of inflammation in the ankle joint, into which 8C1 was previously administered, and has practically no effect on the joint into which the control solution was injected. To assess the degree of inflammation during the 72-hour period after intravenous injection of 8CU using an vernier caliper, the sizes of the ankle joint were determined after 0, 24, 36, 48 and 72 hours after reactivation of arthritis and then another limb was taken for histological examination (for example, inflammation, education pannus, cartilage damage and bone damage).

Results.

The effect of the administration of CTEDED 2.6E / C106bb was evaluated by the development of joint edema during arthritis reactivation. Inhibitors and carriers were administered as a single intravenous injection 24 hours before reactivation of arthritis with 8C1.

KTIECh 2.6E / C106bb showed a statistically significant effectiveness in reducing joint edema in the analysis of deviations (ΑNOUΑ) using the Fisher test (81a1u | e \\ · ®) in all four doses on the second and third days after reactivation of arthritis, and on the first day - in all doses except one (1.5 mg / kg). This reduction in edema was comparable to that obtained with a positive control of CTLECC 4E / C105b, administered daily at a dose of 0.5 mg / kg (i.e., 8 nM), starting from one day before reactivation and ending on the third day after reactivation.

CTDEC. also showed significant effectiveness in the case when the magnitude of the edema for three days was considered as one indicator. The area under the curve (AIS) reflects the dose-response relationship for all doses (see Fig. 9, where CTEC 2.6E / C106b is designated CTECD 2.6Т, and CTEC 4I / C105b CTEC 4 4).

In this model system, the introduction of kTIEKCH 2.6E / D 105-1-VNRES (33 kD) led to a significant decrease in the ankle width and histological parameters when compared with the control group.

Ό. С-galactosamine / lipopolysaccharide model.

The model with Ό-galactosamine (I-Ca1IN ₂ ) / lipopolysaccharide (L8) (Pagte1u e1 a1., (1993), Kirga) is a highly TIE-a-dependent animal mortality model ίη у1уо. In addition, autoimmune mice MKE1rg / 1rr have been shown to be extremely sensitive to LB8 or 8E induced TDE-a (MoshIkh e1 a1., (1995), 1. ^ typ., 155: 4829-4837).

Protocol.

After fasting overnight, the following pharmacological reagents were intravenously administered to 6-8 week-old female MKE-1rg / 1rg mice (1askbop Labogogu, Vag Nagog, ME): 25 mg I-Ca1IN ₂ (81dta Syetua1 Co., 8. suspended in Hank's balanced saline solution (Clbco Eaboguepec, Sch., Cgapb Ilapb, NΥ) (50 mg / ml), lipopolysaccharide (L8) from E.co11 serotype 0127: B8 (81dta Syetua1 Co., 8ΐ. bosh, MO) sterile, endotoxin-free phosphate buffered saline (PB8) (50 mg / mouse) or 8EB (ToxW TesNpokschek, 8agako! a, EB) in physiological saline (50 mg / mouse). Various forms of kTDEI were administered in two serial dilutions (dosage expressed in mg / kg) to obtain EB ₅₀ curves. which were built with the help of the statistical program for Masi'ozi (81aMe ^ ®), Moip! at U1e \\ '. CA. Mortality was assessed at +48 h after infection.

Results.

As can be seen from the table below. 4, when 3ΤΝΡΚ-Ι 2.6B / C106bb was administered as described above, 1 hour before the administration of RHL / O-CaSH. EO ₅₀ (i.e., a 3ΤΝΡΚ-Ι 2.6B / C106bb dose necessary for 50% protection) after 48 hours was ~ 50 μg / kg (Ν = 8 individual mice). Compared with 3ΤΝΡΚ-Ι 4B / C105bb, there was no significant (P> 0.05) distinguishable difference in the ability of this form to reduce mortality (EB ₅₀ = ~ 50 μg / kg; Ν = 8 individual mice).

Table 4

Comparison of 8ΤΝΡΚ and optimized truncated forms of 8ΤΝΡΚ on the BRZ / R-SaGYL model;

Agent ^E O100 Εϋ50 8ΤΝΡΚ-Ι 41) С105с1Ь ~ 100 mcg / kg ~ 50 mcg / kg 8ΤΝΡΚ-Ι 2.61) С106с1Ь ~ 100 mcg / kg ~ 50 mcg / kg 8ΤΝΡΚ-Ι 2.6Ώ / Ν105-! ViRES (33 cd) ~ 2 mg / kg ~ 400 mcg / kg 8ΤΝΡΚ-Ι 2.6Ώ / Ν105 -! - MERES (20 kD) ~ 800-1000 mcg / kg ~ 1 mg / kg 8ΤΝΡΚ-Ι 2.6Ώ / Ν105 -! - MERES (20 cd branched) 2 mg / kg ~ 1-1.5 mg / kg 8ΤΝΡΚ-Ι 2.6Ώ / Ν105 -! - MERES (40 cd branched) 1.5 mg / kg ~ 1 mg / kg

The data presented indicate that 3ΤΝΡΚ-Ι 2.6B / C106bЬ has a similar activity with 3ΤΝΡΚ-Ι 4B / C105bЬ, but 3ΤΝΡΚ-Ι 2.6P / Ш05 -! - ViREC (33 kD) was less active in this model system with the value EB ₅₀ ~ 400 mcg / kg; Ν = 8 individual mice. In addition, the activity of 3ΤΝΡΚ-Ι 2.6Ό / Ν105-! MePEC (20 kD branched) and 3ΤΝΡΚ-Ι 2.6Ό / Ν 105-1-MePEC (40 kD branched) was lower in this model system.

E. Model adjuvant-induced arthritis.

Adjuvant-induced rheumatoid arthritis in rats has much in common with human rheumatoid arthritis. The purpose of this experiment was to show that the systematic administration of truncated 3–3 has a positive effect on the pathogenesis of arthritis induced by adjuvant in mice.

Protocol.

Male Lewis rats (5–7 in the group) (Sjag1e8 P | Б baогаa! Og1ez, Ec, \ 11sh1pd! Op, MA) weighing at least 200 g were implanted with subcutaneous catheters and allowed to get used to them for several days. Then the animals were placed in special cages for infusion and allowed to get used for a week before starting the infusion.

On day 0, 100 μl of Freund's complete adjuvant (81dta Syet1ca1 Co., 81. Bosch, MO) was added to all rats, to which was added a synthetic adjuvant, Ν, Ν-dioctyldecyldecyl-Y ', M-bis (2-hydroxyethyl) propanediamine, 50 mg / ml. On day 8, various groups of rats were administered by continuous subcutaneous infusion of 3ΤΝΡΚ-Ι 4ΙΜΊ05 and 8ΤΝΡΚ-Ι 2.6Ό / Ν105.

The results are shown in table. 5.

Table 5

Adjuvant-induced arthritis

Compound Dose, mg / kg / day AiS% (% suppression) Paw weight (% suppression) Inflammation (% suppression) Bone resorption (% suppression) Experience # 1 8ΤΝΡΚ-Ι 4B / S105 5 61 46 37 89 one 49 45 26 855 0.2 33 40 14 34 8ΤΝΡΚ-Ι 2.6Ώ / Ν105 one 55 53 33 51 Experience # 2 8ΤΝΡΚ-Ι 2.6Ώ / Ν105 5 42 ΝΏ nineteen 67 one 38 ΝΏ thirteen 49 Experience # 3 8ΤΝΡΚ-Ι 2.6Ώ / Ν105- MERES (33 cD) nine fifty 40 thirteen 27 3 35 34 nine 22 one 36 thirty 0 0 8ΤΝΡΚ-Ι 2.6Ώ / Ν105- MERES (33 cD) nine 43 37 3 38 33 one 24 twenty

Surprisingly, 3ΤΝΡΚ-Ι 2.6Ό / Ν105ViRES (33 kD) and 3ΤΝΡΚ-Ι 4B / C105bб showed similar anti-arthritis activity when tested on the adjuvant-induced arthritis model in Lewis rats, while 3ΤΝΡΚ-Ι 4B / C105bЬ was more effective in studies on cytotoxicity of typhoid cells on cells ЕШ-164 and Б929, as well as when testing on the L8 / CaS model system.

E. Collagen-induced arthritis model.

Arthritis in rats induced by type ΙΙ collagen has much in common with rheumatoid arthritis in humans. The purpose of this experiment was to show that the systematic administration of truncated kT # KK has a positive effect on the pathogenesis of collagen-induced type ΙΙ arthritis in rats and mice.

Experience on rats.

Female Lewis rats (Sjag1ek K1uet Laboginaopek, 1ps., ^ 11mshd1op, MA) were implanted with subcutaneous cannulas, which the animals got used to, which made it possible to conduct continuous infusions. Then the animals were immunized with bovine collagen type ΙΙ (81dta C11epis 1 Co., 81. Loshk, MO) in Freund's incomplete adjuvant. On days 13, 14 or 15 after immunization, animals that developed arthritis were randomly divided into groups of 8 animals each. Animals of the experimental groups twice daily were injected intraperitoneally with either buffer or various doses of CTOTI for 7 days, as described in table. 6. Paw inflammation was evaluated daily by determining the diameter of the ankle joints with a caliper. On day + 7, animals were euthanized and paw weight was determined, which served as an indicator of inflammation. Ankles and knee joints were collected for histopathological evaluation of arthritis parameters.

The results are shown in table. 6A.

Table 6A

Collagen-induced arthritis

Compound Dose, mg / kg / day AiS% (% suppression) Paw weight (% suppression) Inflammation (% suppression) Bone resorption (% suppression) Experience # 1 KTIEKL 4B / S105 5 65 81 N0 N0 one 35 34 N0 N0 0.2 nineteen 22 N0 N0 KTIKEL 2.6 | > / N10 " one 39 41 N0 N0 Experience # 2 (mg / kg / day) KTIEK-Ι 2.6V / No. 05MeRES (33 kIa) 3 fifty 60 76 46 CTRL - ^ / N105- MERES (33 kia) 3 47 fifty N0 N0 Experience # 3 (mg / kg / day) KTIEK-Ι 2.6V / No. 05MeRES (33 kVA) nine 25 44 N0 N0 KTIEKL 2.6V / No. 05MeRES (33 kVA) 3 25 37 N0 N0 KTIEK-Ι 2.6V / No. 05MeRES (33 kVA) nine 35 52 N0 N0 KTIEK-Ι 2.6V / No. 05MeRES (33 kVA) 3 35 37 N0 N0

It is noteworthy that in this rat arthritis model for all experimental groups similar results were obtained, for example, the shape of the curve, the percentage (%) of suppression, determined by the area under the curve (AIS), varied between 30-59% and the percentage (%) of suppression , determined by the weight of the paws, ranged from 40-64%. The values for the group that did not receive the drugs were statistically different in this model system from the values obtained for other groups.

Experience on mice.

Male mice IVA / Ι Tsaskkop LabaFpek, Sh., Wag Nagog, ME) were immunized with bovine collagen of type ΙΙ (81dta C11eps1 Co., 81. Loshk, MO) in Freund's incomplete adjuvant. On days 24, 25 and 26 after immunization, animals that developed arthritis were randomly divided into groups of 8 animals each.

Animals of the experimental groups were injected either intraperitoneally with either buffer or kT # K- [2.6I / M05-ViRES (33 kD) for three consecutive days (days +27, +28, +29) twice a day. Inflammation of the paws was evaluated daily, determining the diameter of the ankle joints with a caliper. On day +34, animals were euthanized and paw weight was determined, which served as an indicator of inflammation. Ankles and knee joints were collected for histopathological evaluation of arthritis parameters.

The results are shown in table. 6B.

Table 6B

Collagen-induced arthritis

Compound Dose mg / kg 2Ώ AiS% (% suppression) General histopathology (% inhibition) Experience # 1 k'TNEK.L 4E / S105b 3 49 39

KTOTI-T 4Ώ / Ν105ViRES (33 kD) 3 63 55 Experience # 2 kTNΕK-I 2.6Ώ / Ν105MeRES (33 kD) nine 73 ΝΏ kTNΕK-I 2.6Ώ / Ν105MeRES (33 kD) 3 75 ΝΏ

C. Continuous infusion in a rat model with L8-induced цией-α production.

^ ΗΡΗ-Ι 2.6ШС105с1Ь and кΤNΡВ-I 2.6Э / С106йЬ, ^ ΗΡΗ-Ι 2.6Ό / Ν105 and кΤNΡВ-I 4Ό / Ν105 were administered intravenously using Ake! ™ mini-pumps implanted in the jugular vein (A1x Co., Pa1o A1! O, CA) in accordance with the manufacturer's instructions for 48 hours (1 mg / kg). Serum β-α levels determined with E8A (Sephute, Satbide, MA) were significantly reduced compared with the control 2 hours after the administration of a high dose of EP8.

Example ΙΙΙ. Immunogenicity studies.

Various forms of truncated recombinant soluble KΤNΡB-I were tested for immunogenicity in several animal model systems.

A. Rodents.

^ ΗΡΗ-Ι 2.6P / M05 -! - VTsRES (33 kD) and kΤNΡB-I 4E / C105b (control) were injected subcutaneously (4 mg / kg) on days 1 and 5 of the experiment to female Sprague-Dowley rats (CpagIec Viegk ba, А11ттЦоп, МА) (n = 6-8 / group). Weekly until 21 days, blood samples were taken from the retroorbital sinus. The presence of CM and CS antibodies was determined in the samples.

Table 7

Immunogenicity for Rodents

Time days 0.01 7 14 21 Group + # animal Caption Ι§ω: Caption Ι§ω: Caption Ι "\ Ι Caption Ι "\ Ι TOTAL ^ 2.6Ώ / Ν105- 33 kD RES one neg. 0 0 0 2 neg. 0 0 0 3 neg. 0 0 0 4 neg. 0 0 0 6 neg. 0 0 0 TOTAL ^ 2.6Ώ / Ν105! -VIRES (33 kD) 0 0.00 0.00 0.00 Standard deviation 0 0.00 0.00 0.00 The control 7 0 0 fifty 0 8 0 0 fifty 0 3 0 0 one hundred 0 nine 0 0 0 0 10 0 0 one hundred fifty eleven 0 0 one hundred 0 12 0 0 one hundred 0 thirteen 0 0 0 0 The control 0 0 62.5 6.3

Standard deviation 0 0 15.7 6.3 Time days 0.01 7 14 21 Group + # animal Caption ^ C Caption ^ C Caption ^ C Caption ^ C kTNΕK-I 2.6Ώ / Ν105! -ViRES (33 kD) one neg. neg. 0 0 2 neg. neg. 0 0 3 neg. neg. 0 0 4 neg. neg. 0 0 6 neg. neg. 0 0 kTNΕK-I 2.6Ώ / Ν105! -ViRES (33 kD) 0.00 0.00 0.00 0.00 Standard deviation 0.00 0.00 0.00 0.00 The control 7 neg. neg. 0 200 8 neg. neg. 0 200 nine neg. neg. 0 0 10 neg. neg. 0 eleven neg. neg. 200 400 12 neg. neg. 0 200 thirteen neg. neg. 200 800 14 neg. neg. 0 fifty The control 0 0 fifty 231.3 Standard deviation 0 0 32.7 94.0

As can be seen from the table. 7, with subcutaneous administration of (8C) kΤNΡB-I 4O / C105b at days ⁺ 1 and ⁺ 5, higher titers of anti-kΤNΡB-I antibodies to day ⁺ 21 are noted compared to very low titers of antibodies to kΤNΡB-I 2.6Ό / Ν105- ! ViRES (33 cd). A similar trend in immunogenicity was also observed in rats in which anti-kNV-I CM antibodies appeared by day ⁺ 21. Injections of KΤNΡB-I 2.6Ό / Ν105-! ViRES (33 kD) did not lead to the development of anti-kΤNΡB-I CM antibodies by day ⁺ 21 in rats.

B. Raryu ApiK.

The task of part A of phase Ι research was to determine the pharmacokinetics and immunogenicity of kΤNΡB-I 4E / C105b (0.2 mg / kg body weight [BT]), kΤNΡB-I 3O / C105b (0.2 mg / kg BT) or kΤNΡB-I 2.6E / C105b (0.2 mg / kg BT), respectively, when administered twice to healthy baboons with an interval of 21 days.

Part Ι of the study was divided into two phases. The task of phase A of part Ι was to determine the pharmacokinetics and immunogenicity of various kΤNΤB-I constructs in healthy baboons in response to two injections. Twelve baboons were divided into three groups. Under anesthesia, animals in each group received 0.2 mg / kg BT ^ ΗΡΗ-Ι 4O / C105b, kΤNΡB-I 3E / C105b or kΤNΡB-I 2.6P / C105b. Three baboons were used in each experiment. After 21 days, the animals received a second identical intravenous injection of protein and the animals were observed for another 21 days. Pharmacokinetic parameters and immunogenicity were determined at the time intervals listed below.

The phase task In part Ι of this study was to evaluate the effectiveness of these drugs when tested on a well-developed model of ΤΝΕα-caused mortality (Ekra! E! A1., 1. 8gd. Vek., 59: 153-158, 1995). The lethal bacteremia of E. soi was induced in 16 animals, divided into groups of four animals each, by the introduction of 5-10 x 10 ¹⁰ CFU / kg of live E. soi cells.

The placebo group served as a control for those groups of baboons who were given intravenously κΤΝΕΒ-Ι 4E / C105bb (0.2 mg / kg body weight [BT]), κΤΝΕΒ-Ι 3P / C105bb (0.2 mg / kg BT) or κΤΝΕΒ-Ι 2.6P / C105bb (0.2 mg / kg BT) or κΤΝΕΒ-Ι 2.6ShC105 bb (1 mg / kg BT).

In the implementation of both phases of part I, we used young males and females Raryu api1k (6-11 kg) (B1oteb1ca1 Vekeagsy Eoipbyop, 8ap Apyusho, Z), who were not given food during the night before the experiment. Animals were anesthetized with ketamine (10 mg / kg, intramuscularly) and a cannula was inserted into the cranial vein. Anesthesia was supported by the initial administration of sodium pentobarbital at a dose of up to 35 mg / kg and subsequent repeated injections of sodium pentobarbital at a dose of 3-5 mg / kg / h. Patency of the upper respiratory tract was provided by the introduction of an endotracheal tube, which allowed the animals to maintain involuntary breathing. A catheter was inserted into the femoral artery, which made it possible to obtain repeated samples of arterial blood, as well as to constantly monitor the heart rate and average blood pressure values using a heart monitor for anesthesia Ea! Axore 2000 Schiaxore, 8a Apyusho, ΤΧ). E ^ ΤA or heparin was added to arterial blood samples collected at regular intervals to prevent clotting and immediately cooled on ice. The plasma fraction was obtained by centrifugation at 4 ° C and then stored at -70 ° C. The temperature was monitored using a rectal thermometer. To control urine separation and creatinine clearance, a Fowley catheter was placed. Hemodynamic parameters were monitored every fifteen minutes. All animals were injected with 0.9% sodium chloride (4 mg / kg) through an intravenous dropper. In phase B experiments, animals received additional fluid (10 ml / kg every 15 min) if two of the following physiological parameters coincided: 1) the average blood pressure dropped by more than 30%; 2) heart rate increased by more than 30%, and 3) urine separation decreased to <1ml / kg / h. After taking the initial blood sample and waiting about 1 h, which is necessary for establishing equilibrium, the introduction of proteins began.

In phase A of part Ι of the study, recombinant proteins were introduced through a catheter into the cranial vein and animals were observed for 8 hours, after which all catheters were removed and animals were returned to the cells on day 21. After 24 and 48 hours, as well as on days 3, 5, 8, 11, 16 and 21, the animals were rapidly anesthetized with an intramuscular injection of ketamine (10 mg / kg) and venous blood samples were taken. On day 21, the animals were again anesthetized, a second injection of protein was made, and the whole procedure was repeated according to the same schedule for another 21 days, when the animals were euthanized.

In the phase In part Ι of the study, one hour before the introduction of E. coli cells, four randomly selected animals were given either a placebo or one of the above constructs. Animals were observed for 8 hours, after which all catheters were removed and animals were returned to the cells. The monkeys developed bacteremia. Animals with excessive discomfort were euthanized. Excessive discomfort was determined according to M. SIS as: 1) inability to maintain a sitting or standing position for 12 hours; 2) inability to take water or food for 12 hours; 3) uncontrolled bleeding from the injection site of the catheter; or 4) lack of response to external stimuli. Venous blood samples were obtained at terms -1, 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 24, 48 hours and 3, 5, 8, 11, 16 and 21 days. On day 21, the surviving animals were euthanized.

The presence of antibodies to the introduced recombinant proteins in Raryu was determined using a sandwich-E8A. Briefly, κΤΝΕΒ-ты constructs were adsorbed on E8A plates (1 μg / ml), and then diluted (from 1:50 to 1: 100.00) baboons plasma (100 μl) was added. After washing the samples, horseradish peroxidase conjugate (No. Β) with protein A (0.5 μg / ml) was added and the test was visualized using TMB.

Results (part Ι).

In three constructs, plasma half-lives varied significantly. Using a model-independent method, excretion curves were determined, and the apparent plasma half-life was determined between 8 and 172 hours. Among intact animals, the largest plasma half-lives were in baboons treated with a construct with 4.0 domains (29 hours), and consistently decreased in baboons treated with κΤΝΕΒ-Ι 3P / C105bb (24.7 h) and κΤΝΕΒ-Ι 2.6O / C105bb (21.5 h). The difference, although statistically significant, was only 26%.

Unexpectedly, after the second administration of the corresponding proteins to the baboons, the half-life in the plasma became much shorter, which indicated a faster clearance. This reduction in half-life was most pronounced among baboons treated with κΤΝΡΚ-Ι 4E / C105b, when this period was reduced by 48% (p <0.01) [Fig. 10]. The reduction in half-life was intermediate in baboons treated with κΤΝΡΚ-ΤΝΡΚ 3I / C105bb (31%) [Fig. 11], and the lowest in animals receiving κ-Ι 2.6O / C105bb (14%) [Fig. 12]. The reduction in half-life was not statistically distinguishable in baboons treated with κΤΝΡΚ-Ι 2.6I / C105b.

All drugs were immunogenic for baboons. However, the frequency of immunogenicity was highest in baboons treated with κ обработ-Ι 4E / C105b, intermediate in animals treated with κΤΝΡΚ-ΤΝΡΚ 3O / C105b and the lowest in animals treated with κΤΝΡΚ-Ι 2.6I / C105b (Table 8).

Table 8

Peak Antibody Responses ¹

First 21 days Second 21 days Average 25% - 75% Average 25% - 75% κΤΝΡΚ-Ι 4E / C105bb (n = 4) 3.20 3.20 3.20 3.95 3.50 4.40 κΤΝΡΚ-Ι 3E / C105bb (n = 4) 1.60 0.00 3.65 3.50 1.30 4.75 κΤΝΡΚ-Ι 2.6E / C105 bb (n = 4) 0.00 * 0.00 1.75 1.45 0.00 3.50

/ Logarithmic scale (the reciprocal of the plasma dilution necessary to obtain the half-maximum absorption in the sandwich-EKA; see Experimental methods) * p = 0.056, according to the two-parameter test ΑNΟVΑ Kgivka1 - \\ 'a11ικ (the values of the transformed 1od do not meet the criteria of normality).

The antibody response usually developed by the eighth day after the introduction of the constructs and persisted for 21 days of the study. In addition, the antibody response became stronger as a result of a second injection of protein constructs.

Antibodies were detected in all four baboons receiving κΤΝΡΚ-Ι 4I / C105bb. In addition, antibodies were detected in two of the four animals treated with κΤΝΡΚ-Ι 3I / C105bЬ, and antibodies were also detected in one of the four baboons treated with κΤΝΡΚ-Ι 2.6I / C105bЬ. Analysis using the ΑNΟVΑ program for Ι <Γΐ.ικ1 <; · ι1- \ ν; · ι11ίκ showed that the amplification of the antibody response (transformed 1ode) varies significantly in the three groups depending on time (p <0.05). The analysis of rom-Nos suggested that a significant difference in antibody responses is fundamental between animals receiving κΤΝΡΚ-4O / C105b and κΤΝΡΚ-Ι 2.6I / C105b, with intermediate (and non-significant) responses in animals receiving κΤΝΡΚ-Ι 3I / C105bb.

In two studies conducted on day 21 (p <0.01), a relationship was observed between the appearance of antibodies and a change in clearance. Not surprisingly, in those animals that developed a strong antibody response after the first administration, protein was excreted faster after the second administration. Changes in clearance between the first and second administrations were compared on animals in which the antibody response developed (n = 7) and on those in which it did not develop (n = 5) (Fig. 13).

Antibodies found in the plasma of individual baboons were tested for direct cytotoxicity using ME180 cells and for their neutralizing ability in the test on L-929 cells. None of the antibodies formed in response to the introduction of these three constructs were neither cytotoxic nor neutralizing.

In the phase 1 of part A of the study on baboons, those animals that showed the highest antibody response also showed the most rapid increase in clearance of constructs after the second injection. Thus, the results show that antibody response can reduce the biological half-life and, therefore, therapeutic efficacy of constructs and therefore dose adjustments may be required. However, there were no adverse clinical reactions to the presence of antibodies when the constructs were administered a second time. Thus, therapeutic efforts aimed at modifying such constructs in order to reduce their immunogenicity without significantly affecting the half-life or effectiveness should, first of all, aim to reduce the need for increasing the dose, and not the risk of adverse reactions.

Results. Part 1. Phase B.

Finally, on intact baboons, all three constructs were approximately equally effective in preventing cytokine-induced damage after E.co11 bacteremia when administered at a dose of 1.0 mg / kg Ε \ ν. One of the 4 baboons given a placebo survived; 4 out of 4 baboons survived who received κΤΝΡΚ-И 4I / C105bb or κΤΝΡΚ-Ι 3I / C105b; and, accordingly, 3 out of 4 baboons who received the construct κΤΝΡΚ-И 2.6I / C105b survived. All three constructs prevented the manifestation of the biological activity of ΤΝΡα and had an increased neutralizing ability.

Part ΙΙ.

The specific task of part ΙΙ of this study in baboons was to answer the question of whether repeated treatment of animals (for example, 3 separate injections) with different κΤΝΡΚ-тами constructs leads to subsequent immunogenicity and a decrease in half-life. Additionally, this study was aimed at comparing the immunogenicity and pharmacokinetics of several κΤΝΡΚ-конструк constructs, including κΤΝΡΚ-Ι 2.6I / C105bб, κΤΝΡΚ-Ι 4I / C105bЬ, κΤΝΡΚ-Ι 2.6Ω / Ν 105-1-VnREO (33 kD) and κΤΝΡΚ -Ι

4Ω / Ν 105-1-ViRES (33 kD). Finally, this study was aimed at assessing the clinical significance of the antibody response and altered clearance in the subsequent response to ΤΝΕα-caused lesions (E. soy bacteremia).

On days 0, 21, and 42, baboons were injected intravenously with 0.2 mg / kg of various constructs κΤNΕΡ-I 4E / С 105бЬ, к'1'ΝΕΡ-Ι 2.60 / С 105бЬ, KUMERL 2.6Β / Ν'1 (В-вВпРРС ( 33 kD) or ^ ΝΕΡ-Ι 4Ό / Ν 105-1-ViRES (33 kD) respectively). On day ⁺ 63, the baboons were given 2 mg / kg B \ U of the corresponding construct. On day ⁺ 65 (i.e., 48 hours later), the baboons were given a lethal dose of E. soi, as described above in part части. The main observations obtained in part свод are as follows.

Results (part ΙΙ).

In general, KEKEK ^ 4Ν / Ν 105-1-ViRES (33 kD) and ^ ΝΕΡ-Ι 2.61) / \ 105-1-ViRES1 (33 kD) had a longer half-life compared to ΜΝΕ ^ -Ι 4Э / С105бЬ and κΤNΕΡ-I 2.6E / C105bb in intact animals, regardless of the number of domains. The half-life ranged from 30-35 hours for mono-RESOLVED forms of κΤNΕΡ-I to 10-20 hours for dimeric RES forms. In addition, κΤNΕΡ-I 4Ό / Ν105-1

ViRES (33 kD) and ^ ΝΕΡ-Ι 4P / С105bi had a longer half-life compared to κΤNΕΡ-I 2.6Ό / Ν 105-1-ViRES (33 kD) and κΤNΕΡ-I 2.6E / C105bb in intact animals.

κΤNΕΡ-I 412 / С'105с1Ь and κΤNΕΡ-I

2.6E / C105bb also turned out to be immunogenic with a slight tendency towards a decrease in immunogenicity in ^ ΝΕΡ-Ι 2.6E / C105bb. However, only ^ ΝΕΡ-Ι 4E / C105bb showed reduced clearance with repeated injections. Neither ^ ΝΕΡ-Ι 4Ό / Ν 105-1-ViRES (33 kD) nor 2.6Ό / Ν105-1 ViRES (33 kD) were antigenic, and their clearance rates did not change significantly upon repeated administration.

The sera obtained from each of the baboons (Ν = 3), which were given various compounds, on days ⁺ 21, ⁺ 42 and ⁺ 61, were tested ίη νίίτο for immunoreactivity (E8A sandwich) using various constructs as an adsorbed antigen. For example, serum obtained on day ⁺ 21 (Table 9) from a baboon given 2.6Ό / Ν105-1ViREC (33 kEa) did not react with either ^ ΝΕΡ-Ι 4О / С105бЬ, nor with κΤNΕΡ-I 4Ό / Ν105 when these compounds were used as adsorbed antigen in E8A.

Table 9

Antibody response in baboons Ι§Ο (with caption> 1: 400), day 21

The number of animals η = 3 k'HMMM 2.6E / C105aB CTOTE ^ 2.6Ώ / Ν105ΐ-ViRES (33 kD) kTKhT'R-Ι 4Ώ / Ν105ΐ-ViRES (33 kD) κТNΕΡ-I 4Э / С105ΐ-ВИРЕС (33кД) 8ΤΝΕΡ-Ι 4E / С105аЬ k'GHPM 4Ώ / Ν105 ^ ΕΡ-Ι 2.6Ώ / Ν105-1- ViRES (33kD) 3/3 neg 3/3 neg 3/3 neg ^ ΕΡ-Ι 2.6E / С1050Ь 3/3 neg 3/3 neg 3/3 neg ^ ΕΡ-Ι 41) \ 105-1-ViRES (33 kD) 3/3 neg 3/3 neg 3/3 neg ^ ΕΡ-Ι 4E / S105-1- ViRES (33 cd) ^ ΕΡ-Ι 4E / C105 ab 1/3 react. (400) 3/3 neg

An antibody response with a titer> 1: 400 was considered a positive reaction. Data for days ⁺ 42 and ⁺ 61 are given in table. 10 and 11. It is important that a positive reaction of ίη νίΐΓΟ was observed with serum obtained from one of the baboons previously treated with 2.6Ν / Ν105-1ViREC (33 kD) when tested with ^ ΝΕΡ-Ι 4E / C105bb as an adsorbing antigen (table . eleven).

Table 10

An antibody response in the baboons of the Church of God (with a titer of 1: 400), day 42

The number of animals η = 3 8ΤΝΕΡ-Ι 2.6E / C105aB kTKhT'R-Ι 2.6Ώ / Ν105ΐ-ViRES (33 kD) ^ ΕΡ-Ι 4Ώ / Ν105ΐ-ViRES (33 cD) ^ ΕΡ-Ι 4E / S105ΐ-ViRES (33 kD) 8ΤΝΕΡ-Ι 4E / С105аЬ ^ ΕΡ-Ι 4Ώ / Ν105 ^ ΕΡ-Ι 2.6Ώ / Ν105-1- ViRES (33 cd) 3/3 neg 3/3 neg 3/3 neg ^ ΕΡ-Ι 2.6E / C105ab 1/3 react. (1600) 3/3 neg 3/3 neg ^ ΕΡ-Ι 4Ώ / Ν105-1- ViRES (33 cd) 3/3 neg 3/3 neg 3/3 neg k'GMGM 4E / S105-1-ViRES (33 k1) a) ^ ΕΡ-Ι 4E / C105ab 2/3 react. (3200) 2/3 react. (800)

Table 11

Antibody response in 1dC baboons (with titer> 1: 400), day 61

The number of animals n = 3 8ΊΝΓΚ-Ι 2.6E / С1050Ь Sutkl 2.6Ώ / Ν105- 1-ViRES (33kEa) SUTCM 4Ώ / Ν105- 1-ViRES (33kEa) ZYuTMK 4E / S105- 1-ViRES (33kEa) δΙΝΓΚ-Ι 4E / С1050Ь STL 4Ώ / Ν105 ZYuTK-1 2.6Ώ / Ν105-1- ViRES (33 cd) 3/3 neg 1/3 react. (1600) 3/3 neg ZYuTK-1 2.6E / С1050Ь 2/3 react. (204800) 1/3 react. (1600) 1/3 react. (3200) ZYuTK-1 4Ώ / Ν105-1- ViRES (33 cd) 3/3 neg 1/3 react. (6400) 1/3 react. (6400) ZT \ GR-14PS105-1- ViRES (33kea) ZUTKL 4E / S1050b 1/3 react. (6400) 3/3 react. (12800)

In baboons who previously received constructs three times, the effectiveness of the response to ΤΝΕmediated violations was greatest with the introduction of (1) sTnK-1 4E / C105b. (2) zTMRK-Ι 2.6О / С105бЬ, (3) δΙΝΓΚ-Ι 4Ό / Ν105-1 ВиРЕС (33 kD) and (4) 2.6Ό / Ν105-ΐ-ΒυΡΕ6 (33 kD) (which was determined by survival, multisystem disorders organs (M80P), level 1-6 in serum and the number of white blood cells). It must be borne in mind that this experiment did not take into account differences in the TLR-neutralizing activity of various constructs.

S. Chimpanzee.

The purpose of this experiment was to assess the immunogenicity of various forms of zT # B-1, re-introduced chimpanzees intravenously for 1 month. The forms of sZTKP investigated in this experiment were sZTKP 2.6b / C105bb, zT \ £ K-1 4P / C105bb, sZTKP 41) / C'105-1-ViRES (33 kD), zT \ EK-1 ZAO ^ NEM- ViRES (33 kD), zT \ EK-1 4Ό / Ν105-1 ViRES (33 kD). Each group included 3 chimpanzees. The dosage and experimental parameters were as follows: each chimpanzee was injected with a test substance in the form of an intravenous bolus injection at a dose of 0.1 mg / kg (a total of 8 doses). The dose volume varied depending on the concentration of the test substance. On day 0, prior to treatment, a 5 ml serum sample was taken from each animal. Additional serum samples were taken immediately before drug administration on days 7, 14, 21 and 28.

Preliminary immunogenicity data for chimpanzees are presented in table. 12.

Table 12

Antibody titer 1dC (number of chimpanzees)

Day 0 () Day 7 (2 doses) Day 14 (4 doses) Day 21 (6 doses) Day 28 (8 doses) SUTKL 2.6E / С1060Ь 100 (1) 400 (1) 1600 (1) 3200 (1) 800 (1) 3200 (1) SUTCL 41) С105с1Ь 3200 (1) 400 (1) 1600 (1) 800 (2) 12800 (1) ZYuTKL 4P / M05-1-ViRES (33 kD) SUTC 2.61 T ^ SW-ViRES ί (33 kD) ZYuTKL 4P / M05-1-ViRES (33 kD) 200 (1) * 1600 (1) 100 (1) * 400 (1)

* Note: titers were determined using zT \ TP-1 4E / C105bb as an adsorbed antigen

By day ⁺ 28, all animals (N = 3) treated with hYuBT-1 4E / C105b or zyuTBP 2.6O / C105bb gave a positive reaction (judged by the results of EY8A) with the highest titer of 1: 12,800 or 1: 3200, respectively (table . 12). (Note: in this part of the experiment, all immunizing antigens corresponding to the antigens adsorbed on the E18A plates were used). One of the animals treated with sJTWP 4Ω / Ν105-1-ViRES (33 kD) had a positive antibody response as of days ⁺ 21 and ⁺ 28 (Table 12). It is important that not one of the animals treated with sJTKP 4E / C105-1ViRES (33 kD) or 3T \ 1L-1 2.61) / 105-1-VIRES, (33 kD) responded to the development of anti-sZTKP antibodies for the entire experiment (tab. 12).

As described above in the section on experiments on baboons, the sera obtained on day ⁺ 28 from each chimpanzee (N = 3) treated with various forms of sSTLC were tested for immunoreactivity (E8A) with other constructs using forms of sSTK-1 in as an adsorbing antigen. An antibody response with a titer> 1: 400 was considered a positive reaction. It is important that the sera obtained from chimpanzees, which were injected with SZTKP 2.6P / M05-1-ViRES (33 kD), did not react either with zT№B-1 4O / C105b, nor with zTKP 4Ό / Ν105, when these compounds were used as adsorbing antigen in E8A (table. 13).

Table 13

Antibody response in chimpanzees (GdO with titer> 1: 400)

The number of animals n = 3 KTM'K-G 2.6Б / С105бЬ KUTK.-1 2.6Ώ / Ν105! -VIREO (33 cD) 8YUTK.-1 4Ώ / Ν105- ! -VIREO (33 cd) KTM'K-G 4B / S105-! ViREO (33 cD) 8ΤΝΡΡ-Ι 4B / S105b 8ΤΝΡΡ-Ι 4Ώ / Ν105 κΤΝΡΡ-Ι 2.6Ώ / Ν105 -! - ViREO (33 cd) 3/3 neg 3/3 neg 3/3 neg κΤΝΡΈ-Ι 2.6Б / С105бЬ 3/3 react. (3200) 2/3 react. (3200) 1/3 react. (400) κΤΝΡΈ-Ι 4Ώ / Ν105-! ViREO (33 cD) 2/3 react. (1600) 2/3 react. (3200) κΤΝΡΡ-Ι 4B / S105 -! - ViREO (33 cd) 3/3 neg 1/3 react. (400) 3/3 neg κΤΝΡΈ-Ι 4B / S105b 3/3 react. (12800) 3/3 react. (6400)

Similar data were obtained for animals treated with ΚΤΝΡΡ.-Ι 4E / C105b, κΤΝΡΡ-Ι 4P / C105 -! - ViREO (33 kD) or κΤΝΡΡ-Ι 4P7Ы105 -! - ViREO (33 kD) (Table 13).

GU example.

EAE is an acute or chronic renewable inflammatory demyelinating disease of the central nervous system (CNS) resulting from the sensitization of genetically sensitive animals with neuroantigens, such as myelin basic protein (MBP). EAE is well known in the art and is often used as an animal model of acute multiple sclerosis in humans.

Female rats Lewis Zaskkop Lauba! Guard, Wag Sh-Peog, IU) were anesthetized on day 0 and immunized in the paw pads of the left hind limb with 0.1 ml of an emulsion containing basic myelin protein (MBP) in complete Freund's adjuvant diluted with phosphate buffer solution (RVB) and mixed with an equal volume of Freund's complete adjuvant (CΡA) containing 5 mg / ml Mucobac! Erg! The control rats were injected into the paw pads of the left hind limb with 0.1 ml of RVB / СΡА emulsion without MBP.

Clinical evaluation of the disease was based on the usual score system 0-5. The scatter of indicators was as follows: 0, normal; 0, 5, partial loss of tail tone; 1, complete loss of tail tone; 2, trembling of one hind limb; 3 paralysis of both hind limbs; 4, the disease; 5, death. All injections of κΤΝΡΡ-Ι constructs or vehicle were performed at a dose of 1 mg / kg subcutaneously every second day, starting from day 9 after immunization. All animals were slaughtered for 21 days. The results were presented in two forms: the severity of the clinical manifestations versus time and the integral clinical score of each rat during the course of the disease, calculated as the area under the curve of the daily clinical score versus time. The values of the integral clinical score for the experimental groups were statistically processed compared to those for the control group using the Mann-Wheatley test.

In animals that received only the carrier, an outbreak of the disease was observed around the tenth day, the disease peaked by day 16 and then weakened. κΤΝΡΡ-Ι 4b / C106bb alleviated clinical symptoms by approximately 73% when compared with animals receiving only vehicle. BYuTK .-! 4b / C106-1-ViPEC (33 kD) also eased clinical symptoms by approximately 85%. κΤΝΡΈ-Ι 4b / С105-! ViREO (33 kD) and κΤΝΡΡ-Ι 2.6Ό / Ν 105 -! - ViREO (33 kD) had approximately the same ability to alleviate clinical symptoms (64 and 57%, respectively).

In conclusion, it should be noted that truncated κΤΝΡΒκ are effective in attenuating individual clinical manifestations when tested in this animal model of multiple sclerosis.

The scope of the present invention is not limited to the described variants of its implementation, which illustrate certain aspects of the invention. Various modifications of the invention, in addition to the above, will be apparent to those skilled in the art from the foregoing description.

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EASTOYA (Theirs)

CHMPVEA OG ZEOCELSESIS: 81 (11) ITEM ΤΥΡΕ: ρΕΟΕβΙη (x!)

SOYAEZRESYOESB AOOYEZZ:

(A) (B) (C) (O) (B) (D)

AOOYAEZZBE: ΑΜ6ΕΝ HYS.

ZTYABBT: 1340 R «Ηβνίΐΐιηά Οτίνβ SHTT: TLoizapb Oakz

ZTATB: SaHhEogaha

SSRNTYAT: from

ΖΧΡ: 91320-1789 (ν)

WATCHING YABAOAVB GOYAM:

(A) (B) (C) (O)

MERKHRM ΤΥΡΕ: Είορργ Ohzk SSMROTEYA: HVM RS with a good OREAATIPZ 3Υ3ΤΕΜ: RS-ROZ / MZ-RO5

ZOGTNAYAB: RasvPEXH KeHease »1.0, Wagshop» 1.30 (νχ)

DEPARTMENT OF ARRXHSAT1OY ΟΑΤΑ: (Α) ΑΡΡΡΙΟΑΤΙΟΝ YTSMVEYA: <h) ghyyas πάτε: (ο ορα55: γχοατιον;

(νϋ)

RYAHA ARRHSATHOY SAHA:

(A) ΑΡΡΡΙΟΑΤΙΟΝ {FRUIT: NW 60/021, 443 (B) GHXXOATE: 09-ΛΊ.-Χ996 (νίί)

RYACHOY ARRRHSATHOM RATA:

(A) ARRYSATHOY KCMVEY: 03 60/032, 534 (B) GHYMO OATE: 06-OEC-1996 (νϋ) RYYA ARRYSATHOY RATH:

(A) ARRYSAT10Y (FRUIT: OZ 60 / 037,737 (B) GHHHNS RATE: 23-1ΑΝ-1997

Avr Star Ya1 Suz Reo C1a C1u Bz Tug Xxx H1z Reo 31a Azp Azp Star one 5 10 fifteen eleven· Suz Suz Tht Bz Suz H13 Bz 61y Te Tug Bie Tug Azp Azr Suz twenty 25 thirty Rgo <Zhu Reo <31u 61p lar Tht Azr Suz Agd O1i Suz 31and Star SHU Zeg 35 40 45 RPV Tht Aha Head 01and Azp Nhz Bie AED H13 Suz Bie Star Suz Zeg Bz fifty 55 60 Suz Agd love hee Willow woo C1p Wah Zhi XX Zoe Star Suz Tht Wah AZR 65 70 75 80 Agd Aar YOU νβΐ Suz woo Suz Azd Bz Azp Zhp Tug Agd Nhz Tug Tgr 85 90 95 8 "e 01c Azp Bvi Pb <51p Suz Pb Azp Suz Star Bie Suz Bvi Azp SHU one hundred 105 BY YOU Ya1 Nhz Bvi Star Suz O1p (ZHi Bz OHP Azp Te Wah Suz Te Suz 115 120 X25 Nhz A1a <31u Pb Pb Bvi Ahd Zhi Azp <31and Suz Wah Zeg Suz Star Azp 130 135 140 Suz Bz Bz Star Bvi hee suz Tht Bz Bie Suz Bie Reo (ZHp 11th SHi

ΙνϊίΙ RYACHOY ΑΡΡΙΧΟΑΤΙΟΝ RATA:

(A) ARRHSATHSI {FRUIT: OZ 60 / 039,314 (B) ΓΙΡΧΝΟ OATE: 07-GEZ-1997 (νϋ) RYOCHA ARRH.XSATHSN RATA:

(L) ARR1HSATHOY NRMVBYA: РЗ 60 / 039,792 (В) ГХЬХМв RATE: 04-MAY-1997

145 150 153 160 Azp (2) ΧΝΓΟΒΜΑΤΙΟΝ GOA 5 £ 0 ΙΟ N0: 3:

(νίϋ> ΑΤΤΟΒΝΕΥ / ΑβΕΝΤ ΙΝΕΟΡΜΑΤΣΟΝ:

(A) (B) ЯБ8Х5ТЯАТХ0Н (FRUIT:

(C)

NAMB: ΖίηύΓίοΧ, Tbotaz 0.

32,185 YABTEYEKSB / OSSKBT MTsMVEYA: A-415E

(1) 5E0REMCE SNALSTEKHZT1C5: (Α) 1ΕΝ6ΤΗ: 332 аз apt Х з з (B) ΤΥΡΕ: 11 1 1s ac!

(Α) ΝΑΜΧ / ΚΕΥ: POP (B) ОOSATHOY: 4..324 (χί) REARCH RESPONSE: ZEO ΙΟ N0: 3

SAT ATS САС forest OTT shh STS CAA SSA AAA TAS Automatic telephone exchange САС COT SAL AAT 48 Month LZr Star Ya1 Suz Reo Zhp 31u Roose Tue Not a ± s REO SHP AZP one 5 10 fifteen LAT TSS ATT shh THAT ACC AAS Tss САС AAA WASP lee TAS TTZ TAS LAT 6

Azp Zeg Nez Suz Suz Thye Rose Suz Nhz Ruz Skhu Tbg Tug Rvi Tug Azp

25 30

SAS TOT SSA OSS SSS SSS SSS SAS <5AT SAS SAS TSS SSS SAS TOT SAS SSS144

Azr Suz Reo C1u Rgo C1u SHP Azr Thier Azr Suz Agd C1i Suz C1i Zeg

4045

SSS TSS TTS ACC SST TSA SAA AAS AAS SAS STS ASA SAS TSS STS ASC TSS192

C1u Zeg Rbe Thg A1a Zeg SXi Azp Nhz Rei AED Nhz Suz Rvi ZegSuz

5560

TSS ALA TSS SSA AAS SAA ATS SST SAS STS (CJSC ATS TOT TOT TSS ASA240

Zve Ruz Suz Ahd Ruz SHi Mes C1u C1n Wah Xi Not Zeg Zeg SuzTg

7075

STS SAS SSS SAS SSS ATS STS TOT SSS TSS SSS AAS AAS AAS SAS TAS SSS SAT 288

Uah Azr Aed Azr Thye Uah Suz S1u Suz Asd Ruz Azp S1p Tut Agd Nkhz

80 85 90 95 Tat TSS LOT CAA LAS STT TTS SAS TSS TTS TSS TSA TASZATSS Tug Tgr ZE ZHi Azp Rei Rbe 81p Suz Rbe Souz * one hundred 105 (2) ΣΝΕΟΒΜΑΤΙΟΝ GOA ZEO ΙΟ N0: 4: (x) SYAJAASTEYA13THSZ: (L) ΡΕΝ3ΤΗ: 107 atHpo asiz (nine) ΤΥΡΕ: ayaypo asHb (0) Τ0Ρ0Ρ03Υ: XXpeag (I) SEA TORE ΤΥΡΕ: rEOSehp (Xx) Zeosense OESIAHRTHSI: Zeo ΣΟ N0: 4: Month Azr Zeg Uah Suz Reo 81p ZHu Rose Tug Not H1z Rgo SHL Azp Azp one 5 10 fifteen Sse Not Suz Court Tht Rose Souz Nhz Rose C1u Tbg tug Rey Tug Azp Lzr twenty 25 thirty Suz Reo C1u Reo Zhu ZHp Azr Tbg Lzr Suz Agd 61i Suz SHi Zeg C1u 35 40 45 Zeg Rbe Tbg Ah Zeg ZXi Azp NHz Rey Agd Nhz Souz Rey Zeg Suz Star fifty 55 60 Roose Suz Agd Rood Zhi Mee C1u SHXp UaH C1 and Not zeg Zeg Suz TBE Wah 65 70 75 80

101

102

Azr Ahd Azr Tne ν · 1 Suz C1u Suz Ahd University of Azp ZHp Tug Ahd N1z Tug 85 90 95 TER Zeg SHi Aza Vei PH SHp Suz PH Suz one hundred 105 (2) ΧΝΓΟΚΜΑΤΙΟΝ GOA Zeo XB N0: 5:

(1) GREECE ZEOCEXZT1SZ:

(A) ΒΕΝΒΤΗ: 339 Base ra1hz (B) ΤΥΡΕ: Х «1 1s ac14 (C) 8ΤΑΑΗΟΕΟΝΪ53: bkpomp (B) Τ0Ρ0Β03Υ: unicomp <ίί) MORE ABOUT: SOMA (1x> location:

(Λ) ΝΑΜΕ / ΚΕΥ: SEZ (B) ΙΛΟΑΤΙΟΝ: 4..333 (χί) δΒΟυΕΝεε OEZSVHRTHOI: 5ЕО 10 N0: 5:

SAT ATS SAS m «e Azr 1 ASS STT TSS SSS SAA SSA AAA TAT ATS SAS SST SAA AAT Star Wah suz 5 REO SHP 31u University Tug 10 XX · Nhz Rgo ZHp Azp fifteen LAT thesis ATT Tss TST ACC AAS Tss САС AAA SSA ACC TAS TTS TAS AAT AZP 8 "E XI Suz Suz Tne University Suz H13 University SHU TNG Tug Vei Tug Azp twenty 25 thirty САС TST SSA sss SSB sowing САС SAT NEA YOU Tss Gas station ZAZ tzt ZAZ ACC Azr Suz Reo SHU Rgo SHU 61p Azr TNG Azr Suz Agd SHi Suz Zhi Zeg 35 40 45 STS shh TTS ACC FTA TSA CAA AAS САС STS AZA САС Tss STS Gas station Tss SHU Zeg Pb Tle Aha Star SHi Azp I13 Bie Agd Naz Suz Vei Zeg Suz fifty 55 60 Tss AAA Tss S6A AAS CAA Automatic telephone exchange FTA SAZ STS САС Automatic telephone exchange TST TST Tss ASA Zeg University Suz Ahd University SHi Men SHU SHP Ya1 31-4 IX 5·: Zeg Suz TNG 65 70 75 STS САС STS САС ACC STS TST sss Tss forest ALZ LAS SAZ TAS STS SAT Ya1 Azr Ahd Azr Tht wah Suz SHU Suz Agd University Azp Zhp Tug Agd H13 80 85 90 95

TAT TOO AST SAA AAS STT TTS SAO TSS TTS AAT TSS TST STO TAL Tug Tgr Zeg S1i Azp Ley RNe Skhp Suz RN · Azp Suz Zeg Lye *

100 103 110

ALZSTT (2) ΧΝΓΟΚΜΑΤΙΟΝ GOA ZEO XO N0: 6:

(1} 5E0CENTER5 SNAVASTA15TXS8:

(A) VIJUTN: 110 aaXpo ac14z (B) ΤΥΡΕ: ab1po ac! H (B) Τ0Γ0Β06Υ: X1peag

144

192

240

288

333

339

YOU Azr TST SSA SSS SSV VVS SAV SAT ASV VAS TSS ACC VAS TST VAS ABC Suz Rgo C1u 35 REO SCHU SHP Azr Thier Azr 40 Suz Ahd SHi Suz SHi 45 Zeg 93C Tss TTS ACC SST TSA CAA AAS SAS STS ASA SAS TSS STS ABC Tss Zhu Zeg Ri tne AX Zev SHI Azp Nhz Wei Agd H1z Suz Vei zeg Suz fifty 55 60 Tss AAA TSS SSA AAS VAL ATS HERE SAV STS ZAV ATS TST tst shh ASA Zeg University Suz Ahd University of Xi Men C1u SHP Wah SHi X1e Zev Zeg Souz Tne 65 70 75 STS САС Pops CAC ACC STS TST FTA TCC ABC AAS AAS SAZ TAS SVZ SAT 7 * 1 Azr Ahd Azr Thr Wah Suz Zhu Suz Agde University Azp VHp Tug Ahd H13 80 85 90 95 Tat Tss AST ZAD LAS STT TTS САС TSS TTS AAT TAD TA66VATSS Tug Tgr Zeg 81i Alp Ley Rye Zhp Suz Rye Azp one hundred 105 (2) ΧΝΓΟΚΜΑΤΙΟΝ GOA 5E0 GO N0: 8; (1) SNOWAASTEECHZTHHSZ (BUT) ΒΕΝ3ΤΗ: 107 ao! By as! 4z (IN) ΤΥΡΕ: ouch! By as1C (ABOUT) ΤΟΡΟΒΟΟΥ: Npeag (11) MOVESTVE ΤΥΡΕ: rgosekhp. (χίΐ βεοϋΕΝΟΒ : 8E0 GO N0: 8: men Azr Zeg U * 1 Suz Reo SHP C1u University of Tug XI · Nkhz Rgo ZHp Azp Azp one 5 10 fifteen Zeg Not Suz Suz Thx University Suz Nhz University of Vkhu Not tug lei Tug Azp Azr twenty 25 thirty Suz Rgo C1u Rgo 81u SHP Azr Tne Azr Souz Avd SHi Suz 81 and Zev VHu 35 40 45 Zeg Pb Tne AXa Zeg SHi Azp Nhz Vei ahd H1z Suz Wei . Zev Suz Zeg fifty 55 60 University Suz Ahd University SHi men 31 y SHP 7a1 CXi IX · 3 "e Zev Suz Tne : Wah 65 70 75 80 Azr Ahd Azr Tne Wah suz zhu  Suz ; Ahd University Azp 31p Tug Ahd Nh: (Tug 85 90 95 Tgr Zeg Zhi Azp Wei Rye ZHp ί Souz ί rne azp one · one hundred 105

(2) ΧΝΓΟΚΜΑΤΙΟΝ GOA ZEO GO N0: 9:

(ί) ΪΕΟϋΒΝΟΞ SYAIASTEYA15T1SZ: (Λ) VIAZTI: 289 азе раххз (B) ΤΥΡΕ: пісхе1с ас! й (C) 3ΤΒΛΝΒΕΒΝΒ53: ипппоип (В) ΤΟΡΟΒ03Υ: ипкпомп (Н) МУСТЕВЕ ΤΥΡΪ: СОМА (1)

(A) ΝΑΜΕ / ΚΕΥ: SHZ (B> LOSATYUN: 4..279

144

192

240

288

333 (11) MOVIEVET ΤΥΡΒ: rgoneXn (χί) ZEOCEISE 0Ε3Ο <ΧΡΤΙ0Ν: ЗЕО 10 N0: 6:

may Azr Zeg 7 * 1 Suz Rgo Zhp Zhu Bz Tug XI H13 gho Zhp Azp Azp one 5 10 fifteen 8 "g XX Suz Suz tng University Suz Nhz Bz SHU Tne Tug Vei Tug Azp Azr twenty 25 thirty Suz Rgo Zhu REO Zhu SHP Azr Tne Azr Suz Agd SHi Suz SHi Zeg Zhu 35 40 45 ZOE PH TNG Aha Zeg SHi AZP Nhz Bie Agd Nhz Suz Vei 8eg Suz 5eg fifty 55 60 Bz Suz Agd love SHi men SHU SHP ν * ι SHi XX Zeg Zeg Suz TNG 7a X 65 70 75 80

Azr Ahd Azr Tne UaH Suz ZHu Suz Lsd University of Azp ZHp Tus Ahd N1z Tug 85 90 95

Thr Zeg C1 and Azp Wei Rie ZHp Suz Rie Azp Suz 3 «e Lye * 100 105 110 (2) ΧΝΓΟΚΜΑΤΙΟΝ GOYA 5E0 ΣΒ N0: 7:

(ί) ZEOOTHSE SNAYAASTEK18THSZ:

A

Ιίί) MOVESTVE ΤΥΡΕ: SOIA (χί) ZBOiVKSV ΟΕδΟΒΧΡΤΧΟΝ: 8Е0 10 N0: 9:

SAT ATS TST ASS AAS TSS SAS AAA SZA ASS TAS TTS TAS AAT VAS TST men X Suz Thr University High School K1z 5 University SHU Tne Tuv 10 Vei Tug Azp Azr Souz fifteen SSA Air force CER 633 SAZ BAT NEA YOU tss Gas station САС TZT SAV Gas station OSS Tss Rgo Zhu Reo VHu VHp AZR Tne Azr Suz Ahd Zhi Suz Zhi Zeg SHU Zeg twenty 25 thirty TTS ACC eating TSA EVIL AAS САС STS Ava САС TZS STS Gas station TZS Tss AAA Pb Te Aha Pharynx SHi AZP H1z Vei Ahd K1z Suz Vei Zeg Suz Pharynx Bz 35 40 45 Tss SOA ALZ SHAFT Automatic telephone exchange 63T SAZ otz ZAZ Automatic telephone exchange TST TST TZS ASA ztz YOU Suz Ahd University Zhi Men Zhu Zhp Wah Zhi Hehe Pharynx Zeg Suz THx Wah Azr fifty 55 60 SVZ YOU ACC STS TZT Air force Tss Gas station AAS AAS SAZ TAS SVZ SAT Tat thesis Ahd Azr Tne ν * 1 Suz 31u Suz Avd University Azp Zhp Tug Agd H13 Tuv Tgr 65 70 75 ABT CAA AAS STT TTS SAZ Tss TTS AAT Tss TST STZ TAA AASSTT Zeg SHi Azp Bie RNe SHP Suz Pb Alp. Suz Zeg Vei 80 85 90

144

192

240

285 (ίχ) ΓΕΛΤϋΗΕ:

ΙΑ) ΝΑΜΕ / ΚΕΥ: POP (3) WATTHHOE: 4..324 (x!) ZEOSEISE ALL WEIGHT: ZE <2 ΣΒ N0: 7:

SAT Automatic telephone exchange YOU ABC STT Tss STS CAA SSA AAA Tat Automatic telephone exchange САС FTA CAA AAT Men Azr Zeg Wah Suz REO Zhp SHU University Tug IX H13 p £ 0 SHP Azp X 5 10 fifteen LAT shh ATT Tss TST ACC AAS Tss САС AAL SSA lee TAS TTS TAS AAT Azp Zeg XX Suz Suz Te University Suz H1z University SHU TNG Tug Vei Τντ Azp twenty 25 thirty

(2) ΙΝΓΟΗΜΑΤΙΟΝ GOA ZEO 10 N0: 10:

(1) ZEOVEYAS SNAYAASTEYA 15THSZ:

(A) ΒΕΝ3ΤΗ: 92 at! As-is (B) ш: аш1по ас14 (О) ΤΟΡΟΒΟΟΥ: Нпэав (11) MOVESTVEST ΤΥΡΕ: rgoneXn (x!) ZEOUEISE OEZESHRTYU: ZEE XO N0: 10:

Mes Suz Tne University of Suz M1a University of ZHu TNG Tug Vei Tug Azp Azr Suz Rgo 15 10 15

61u REO 31u ZHp Azr Tne Azr Suz Agd SHi Suz Zhi Ze: ZHu Zeg PH · 20 25 30

103

104

Bx Aha Zeg SHi Azp Nhz Bie Agd H1z Suz Bie Zeg Suz Zeg Bz Suz 33 40 45 Agd Bz C1 and meer SHU SHP Wah SHi X1e Zeg Zeg Suz Bx Wah Azr Agd fifty 55 60 Azr tg V "! Suz SHU Suz Agd Bz Azp SHP Tug Agd Nhz Tug Tgr Zeg 65 70 75 80 C1 and AZP Bie Pb SHP Suz Pb Azp Suz Zeg Bie •

90 (2) ΧΝΓΟΗΜΑΤΧΟΝ ROYA ZEO Ιϋ N0: 11:

(1) 5ΕΟϋΕΝΟΣ SHAJAASTEYAHZTKhSZ: (Λ) ЕМEMET.CH: 315 Base ra ± gz (B) ΤΥΡΕ: pisiekhs ac1y (C) 3ΤΑΑΝΟΕΟΝΕ33: ipkpomp (O) TOROOST: ipkpomp (11) SESSION ΤΥΡΕ: οΟΝΑ. . (1x)

(A> ΝΑΜΕ / ΧΕΥ: SOZ (B) LOSATHOI: 4..309 (xx) ZEOTEIS OEZSNHRTHOY: ZEO U N0: 11;

SAT ATS Tat Automatic telephone exchange САС FTA CAA AAT AAT Tss ATT Tss TST ACC AAS Tss 48 Month Tug X1e Nhz Rgo SHP Azp AZP Zeg Hehe Suz Suz Bx Bz Suz one 5 10 fifteen CAC AAA WASP ACC TAS TTS TAS AAT САС THAT SSA STS STS STS САС SAT 96

Nhz bz C1y bt Tu; Ley Tu; Azp Azr Suz Rgo C1u Rgo C1u C1p Azr

25 30

ACC CAC TCC ACC CAC TOT CAC ACC CCC TCC TTC ACC CCT TSA CAA AAC144

THx Azr Suz Lhd SHi Suz SHi Zeg ZHu Zeg Rie Thx ThaaXa Zeg C1i Azp

4045

SAS STS ASA SAS TSS STS ASS SSS TSS AAA TSS SSA AAS SAA ATS SOT192

H1z Ley Ahd H1z Suz Ley Zeg Suz Zeg Luz Suz Agd Luz C1 and Ms Skhu

5560

SAZ STS SAS ATS TST TST TSS ASA STS SAS SSS SAS SAS ASS STS TST SSS240

C1n Wah Xi Not Zeg Zeg Suz Thr V ·! Azr Agd Azr Thr V *! Suz CXu

7075

TSS ACC AAS AAS SAS TAS SSS SAT TAT TSS AST SAA AAS STT TTS SAS288

Suz Agd Luz Azp S1n Tug Agd H1z Tgr Zeg S1 and Azp Ley Pye S1n

85 9095

TSS TTS AAT TSS TST STS TAA AASSTT315

Suz Rye Azp Suz run Yee *

100 (2) ΧΝΤΟΡΜΑΤΙΟΝ GOA ZEO HO N0: 12:

(1) ZEOEMSE SNAYAASTEYAHZTHSZ:

(A) EMOTION: 102 ayaypo as1yz (8) ΤΥΡΕ: aah1pa as! 4 (O) TOROOOOOT: 11peah (11) NON-SESSION ΤΥΡΕ: rgoeekhp (x1) ZESEOGGSE OEZESHRTHOYA: ZEO XO N0: 12:

ACC STS Tss TTS ACC FTA TSA CAA AAS САС STS ASA САС Tss STS forest Zeg SHU Zeg Pb Tht A1a Zeg SHi Azp Nhz Bie Ahd H13 Suz Bie Zeg 35 40 45 Tss Tss AAA Tss SSA AAS CAA Automatic telephone exchange FTA САС STS САС Automatic telephone exchange TST TST shh Suz Zeg Bz Suz Agd Bz C1 and Month C1u C1p Wah SHi Hehe Zeg zeg Suz fifty 55 60 ASA STS САС STS САС ACC STS TST STS Tss ACC AAS AAS САС TAS STS Tg Ya1 Avr Ahd Azr Tht 7a1 Suz C1u Suz Agd Bz Azp SHP Tug Agd 65 70 75 SAT Tat Tss AST CAA AAS STT TTS САС Tss TTS AAT Tss TST STS TAA H1z Tug Tgr Zeg SHi Azp Bie Pb SHP Suz Pb Azp Suz Zeg Bie • 80 85 90 95

AASSTT

144

192

240

288

294

(2) ΙΝΓΟΗΜΑΤΙΟΝ GOA ZEO ΙΟ N0: 14: (1) ZEOCENSE SNAYAASTEYAHZTHHSZ: (BUT) LIETN: 95 atpo asHyz (IN) ΤΥΡΕ: ahpo askh4 (ABOUT) TOROYOOT: Ipeag (11) MOSCOW ΤΥΡΕ: rgoeehp (Xx) Zeoselse OEZSNHRTHOI: ZEO HO N0: 14: Mee Zeg Not Zeg Suz YOU SOUZ NHS Bz C1u YOU Tug Bie Tug Azp one 5 10 fifteen AZR Suz Rgo C1u Rgo CXu C1n Azr Thr Azr Suz Ahd SHi Suz C1 and Zeg twenty 25 thirty SHU Zeg Pb Tht Aha Zeg SHi Azp Nhz Bie Agd H13 Suz Bie Zeg Suz 35 40 45 Zeg Bz Suz Agd Bz SHi Mee C1u SHp Wah SHi Not Zeg Zeg Suz YOU fifty 'h ⁵⁵ 60 Ya1 Azr Agd Azr YOU Wa1 Suz C1u Suz Ahd Bz Azp SHP Tukh Ahd Nhz 65 70 75 80 Tug Tgr 5eg SHi Azp Lye Rye S1n Suz Pb Azp Suz Zeg (whether *

90 95 (2) ΧΝΓΟΗΜΑΤΧΟΝ GOA ZEO 10 N0: 15:

(I) SESSION OF SNAYAASTEEKHZTHSZ:

(A) LEFT: 4 AP! By ASHBZ (B) ΤΥΡΕ: AB1B by ASHB (C) WRONG: IKPOMP (O) TOROOOOOT: IKPKOMP (II) MOGIETS ΤΥΡΕ: PRGOSehp (x!) ΕεαυΕΝΟΕ OXECTS: OXECTS:

Aza Zeg Not Souz

her Tug X1e H1z Rgo C1p Azp Azp Zeg Hehe Suz Suz YOU Bz suz Nhz one 5 10 fifteen Bz SHU Bx Tug Bie Tug Azp Azr Suz REO C1u Rgo C1u C1p Azr YOU twenty 25 thirty Azr Suz Agd SHi Suz C1 and Zeg C1u Zeg Pb Tht Aha Zeg SHi Azp H13 33 40 45 Bie Agd nhz Suz Bie Zeg Suz Zeg Bz Suz Agd Bz SHi Meg C1u C1p fifty 55 60 Wah C1 and Hehe Zeg Zeg Suz Tht Ya1 Azr Agd AZR Tht Wah Suz SHU Suz 65 70 75 80 Agd Bz Azp C1p Tug Agd H13 Tug Tgr Zeg C1 and Azp Bie Pb SHP Suz 85 90 95

(2) ΣΝΤΟΒΜΑΤΙΟΜ GOA ZEO HO N0: 16:

(1) ZOOPEXE SNAYAASTEYAHZTHSZ:

(A) EXCEPT: 5 ASHPO ACCOS (B) ΤΥΡΕ: ACCEPA AC! C (C) 3–53: IPCOMP (O) TOROOOOT: IPCOMP (I) MOSTIES ΤΥΡΕ: PROCESSING (X1)

Azp Azp Zeg X1e Souz

5

Rye Azp Souz Zeg Lye

100 (2) ΙΝΓΟΒΜΑΤΙΟΝ GOA ZEO Yu N0: 13:

(ί) ZEOCEMSE SYAJAASTEIA13T1SZ:

(A) Б К СТ К 29: 294 аз ра ра х з (((8) пис: 1 1 1 ас ас ас <<5 <5 (C) HEALTH CARE: к к п омп (: п: к п омп:::: к к п омп (11: ΤΥΡΕ ΤΥΡΕ и и С: SOMA (xx) GEOE:

(A> ΝΑΜΕ / ΚΕΥ: POP (B) СOSATHOY: 4..288 (2) ΙΝΓΟΗΜΑΤΧΟΝ GOY ZEO GO N0: 17:

(1) ZEOCENSE SNAYASTEKHZTKhSZ: (A) EXT: b atkhpo ASKZ (8) ΤΥΡΕ: ALKHPO ASK (C) 5-33: Ukkompom (O) TOROOOT: Ukkompom (11) MOSCEPT EXCEPT EXCEPT thirteen

SAT PBX me one Tss Zeg ATT ACC THAT Suz 5 ACC AAS TCC SAS AAA SSA ACC TAS TTS TAS 48 Hehe Zeg Bk Suz Nhz Bz 10 C1u Tht Tukha lei Tug fifteen AAT САС TST SSA weight STS STS САС SAT ACC САС Tss ACC OJSC TET САС 96 Azp Azr Suz Rgo SHU Rgo SHU C1p Azr Tht Azr Suz Ahd SHi Suz SHi twenty 25 thirty

(х!) ЗЕОБИСЕС ОЕСЯ1РТ1ОЫ: ЗЕО GO N0: 17:

C1p Azp Azp Zeg X1e Suz _h 5 · (2) ΙΝΓΟΗΜΑΤΙΟΝ GOA ZEO GO N0: 18:

(x) ZEOCEISSE SYAAYASTEIA13T1SZ:

(A) EXCEPT: 7 agachpo acxc3 (B) ΤΥΡΕ: at1po ac! 4 (C) 3-33: ipkpomp (O) TOROOOOT: ipkpomp.

(ϋ) MOSCOW ΤΥΡΕ: rgoeekhp (xx)

Rgo C1p Azp Azp Zeg X1e Suz

5

105

106 (2) ΙΝΓΟΒΜΑΤΙΟΝ GOW 5Ε <5 ΙΌ N0: 19:

(ί) 3ΕΟϋΕΝΟΕ СНААСТЕАХЗТ1С5: (Λ) ΕΕΝ6ΤΗ: 8 ахпо ашйз (В) ΤΥΡΕ: аа! по ас1й (С) 3ΤΒΑΝΟΕΟΝΕ33: ипппоип (О) ΤΟΡΟΕΟΟΥ: ипппоип (IX) МОЬЬЕЕ ΤΥΡΕ: ргоЕехп (ЬЕПЕПЕЗЕЗЕПЕЗЕПЕЗЕЗЕПЕЗЕПЕЗЕЗЕЗЕЗЕЗЕЗ) N0: 19

Nkhz Rgo 61p Azp Azp Zeg Ne Suz

5 (2) ΧΝΓΟΒΜΑΤΧΟΝ ROX ZEO GO ΝΟ: 2 £:

(ί) ZEOEISSE SAYAASTEYAHZT1SZ: (Λ) ΙΛΝ6ΤΗ: 15 aphao as £ 0z (B) ΤΥΡΕ: athpo as £ 4 (C) 3ΤΗΑΝΟΕΟΝΕ33; ipkpoip (O) Τ0Ρ0ΙΛ0Υ: ipkpoip (xx) MOSES ΤΥΡΕ: rgosekhp (xx)

Suz Rgo 01p (51y Luz Tug Not Ηίβ Rgo 61p Azp Azp Zeg 11e Suz 15 10 15 (2) ΙΝΕΟΒΜΑΤΙΟΝ GOK 5E0 10 N0: 20:

X N0: 20

11th Nhz Rgo C1p Azp Azp Zeg 11th Suz

5 (2) ΙΝΕΟΒΜΑΤΙΟΝ GO ZEO 10 N0: 27:

(1) ZEOSEIS SNAYASTEY13TKhSZ: (Α) 1ΕΝ6ΤΗ: 16 atpo х х (: МО МО МО МО МО МО МО МО 27 МО :

Уа1 Суз Ρεο О1п С1у Нуз Тус Не Нхз Рго 61п Азп Ап Зег Х1е Суз 15 10 15 (2) ΙΝΕΟΒΜΑΤΙΟΝ GOA ZEO Yu N0: 21:

(1) ZEOTSEYSE SNAYAASTEYA15THS5: (Α) 1ΕΝ6ΤΗ: 10 athpo asHyz (B) ΤΥΡΕ: athpo askhy (C) 3ΤΒΑΝΟΕΟΝΕ35: ipkpoip (O) ΤΟΡΟΕΟΟΥ: ipkpoip (XX) MOESTSE TYPE: _h rgoseHp (χί) ZEOTSEISE OEZSYAHRTYUY: ZEO Yu N0 : 21:

Tug 11th NHC Rgo <31p Azp Azp Zeg 11e Suz 15 10 (2) ΙΝΕΟΒΜΑΤΙΟΝ GOA ZEO 10 N0: 28:

(x) ZEOOEYAS SNAYASTAYA13T1S5:

(A) LEFT: 17 atpo х х з ((B) ат: х п ас х х ((C) 3ΤΒΑΝΟΕΟΝΕ35: ipkpoip (O) ΤΟΡΟΕΟΟΥ: ipkpoip (χί) MOBILE ΤΥΡΕ: rgosekhp (xx) 5 (0υΕΝΟΕ ϋΕδΟΕΙΡΤΙΟΝ: 5Е0 10 N0: 28

Zeg Wa1 Suz Rgo C1g. <31u Luz Tue X1e Nhz Reo C1p Azp Azp Zeg 11e 15 U 15

Suz (2) ΙΝΕΟΒΜΑΤΙΟΝ GOA ZEO 10 N0: 29: (ί) ЗЕОИСИС СЯАЯАСТЕЯ13Т1СЗ:

(A) BJTN: 18 athpo askpz (B) ΤΥΡΕ: atKhpo as! 3 (C) 3ΤΒΑΝ0Ε0ΝΕ33: ipkpoip (O) ΤΟΡΟΙΟβΥ: ipkpoip (xx) MOOESSEE ΤΥΡΕ: prgos »1P (2) ΙΝΓΟΒΜΑΤΧΟΝ GOA ZEO XO N0:

(X) ZEOSEISSE SNAYASTEIA13TKhSZ:

(A) LIOTN: 11 atpo ASKhaz (B) ΤΥΡΕ: atpo ASK (C) 3-33: IKKPOIP (O) ΤΟΡΟΜΟΥ: IKKPOIP (XX) MILLION ΤΥΡΕ: PROSEKP (xX) ZEOTEIS ΟΕ5σΚΙΡΤΙΟΝ: ZEO XO N0

Luz Tuh X1e NHZ Rgo C1p Azp Azp Zeg Ne Suz 13 10 (xx) ZEOSEKSE OEZSAHRTHON: ZEO XO N0: 29:

Azr Zeg ν> 1 Suz Ρεο 61п <31у узз Тух Не Н1з Рго С1п Azp Azp See 15 10 15

Not Souz (2) ΧΝΓΟΗΜΑΤΙΟΝ GOA ZEO 10 N0: 23:

(X) ZEOEXSE SNAYASTEIA 15T1SZ: (Α) ΕΕΝΟΤΗ: 12 atkhlo as! 4z (V) ΤΥΡΕ: atKhpo as! (C> 3ΤΒΑΝΟΕΟΝΕ33: ipkpoip (O) ΤΟΡΟΕΟβΥ: ipkpoip (XX) XO N0: 30:

(x) ZEOCEISE SNAVASTEAHZTHHSZ:

(A) HELP: 4 atpo ac133 (B) ΤΥΡΕ: atpo acx <4 (C) 3-33: ipkpoip (O) ΤΟΡΟΙΟΟΥ: ipkpoip (1x) MOSCOW ΤΥΡΕ: rgosekhp (xx) 3ΕΟϋΕΝΟΕ ΟΕδΟΕΙΡΤΣΟΝ: ZEO 10 N0: 30

Ρίιβ AzpSuz Zeg (XX) ZGAYEESE OEZSVHRTYU: ZEO 10 N0: 23:

С1у Луз Туг 11е НХз Рго С1п Азп Ап Зег 11е Суз

10 (2) ΙΝΕΟΒΜΑΤΙΟΝ GOA ZEO 10 N0: 31:

(x) SESSION SNAYAASTEYA13T1S5: (Α) ΙΕΝΟΤΗ: 5 atpo ASHOZ (B) ΤΥΡΕ: at1po at ASU (C) 5ΤΒΑΝΟΕΟΝΕ35: ipkpoip (O) TOROOOOO? ipkpoip (ϋ) MOBILE ΤΥΡΕ: rgosekhp (2) ΙΝΕΟΒΜΑΤΙΟΝ GOA ZEO 10 N0: 24:

(ί) ZEOOEISSE SNAYASTEYA 15T1SZ:

(A) EXCEPT: 13 atxpo ACCOS (B) ΤΥΡΕ: atXpo ACCS (C) 3ΤΒΛΝ0ε0ΝΕ55: ipcpoip (O) ΤΟΡΟΕΟΟΥ: ipcpoip (xx) MOOSE ΤΥΡΕ: pgosexp (χί) EXCEPT OPERATED: OPEN EXECUTIVE: OPEN

Rye Azp Souz Zeg Lye

5 (хх) ЗЕОЦЕЖЖЕ 0Ε30ΒΙΡΤΣ0Ν: ЗЕО 10 N0: 24:

<31p O1u Buz Tus Ne Nkhz Rgo C1p Azp Azp Zeg X1e Suz

10 (2) ΙΝΕΟΒΜΑΤΙΟΝ HERE ZEO 10 N0: 23:

X N0: 25:

Rgo <31p 61u Euz Tue 11e N1z Ρεο 61p Azp Azp 5eg Not Suz 15 10

107

108 (2)

ΙΝΓΟΒΗΑΤΙΟΝ GOA 5Ε0 W N0: 32:

(ί)

ZEOOEISSE SYAYALSTEIA15T1SZ:

(A) (B) {C) <P>

LIEKOT: 6 APHPO AS1YZ TOUR: aaHpo ASY! ZMYATOMEPYEZZ: Ipkopyp TOROYOSU: Ipkpyop

MAYESHT'E TOUR: rgosekhp (2) ΙΝΓΟΚΗΑΤΧΟΝ goya ЗЕ <2 TO N0: 35:

(1) 5Ε0ϋΕΝθε SNAYAASTEA13TTSZ:

(λ) ΧΕΝΟΤΗ: 235 atHpo ashyz (B) ΤΥΡΕ: as ± in ac1b (O) TOROSST: Npeag (11) MOKHESKHE ΤΥΡΕ: rgosekhp (2) (2) (x!)

Pb

ZEOSEMSE 0Е5СЯ1РТ10Ы: ЗЕО 10 N0: 32:

Azp Souz 5 * ^g Yey Suz

ΧΝΓΟΑΜΑΤΙΟΝ GOA 5E0 10 N0: 33:

(1) (11)

ZEOSENSA SNAYASTEIA15T1SZ:

(A) (B) (C) (O)

SHUTTER: 7 atapo ac16z TOUR: aokhpo as1 ZTIAAIOEOYEZZ: ipkpoip TOROYOSU: ipkpoip

MOON'S TOUR: rgosekhp δΕΟϋΕΝΟΕ 0 ЕЗСЯ1РТ10Ы: ЗЕО Ю N0: 33:

Pb

Azpsuz Zeg Ley Suz Ley

ΙΝΓΟΚΜΑΤΙΟΝ GOA ZEO 10 N0: 34:

(one)

LIST: 705 base of RAKhkhz TOUR: pis1e! S as! D 5ΤΗΑΝΟΕΕΝΕ33: ipkpoip TOROOOOO: ipkpoip

SECURITY ΤΥ? Ε: 'ςβΝΑ <1x)

GEATE:

(A) ΝΑΜΕ / KEU: POPs (B) ΙΧΧΑΤΙΟΝ: 1..705

one.

(HA)

ZEOEEMSE ΟΕδΟΑΙΡΤΙΟΝ: ZEO 10 N0: 34:

AXA 61P WHAAXA REE TH:

01y

ACC

Zeg

ASA TSS SSS Tg Suz Agd

Cts bie

Tss suz

(X1) ZEOOEMSE OEZSAHRTTSY: : ZEO 10 N0: 35: Hei Rho Aha 61p Wah A1a Pb Tht Rgo Tug A1a Rgo SHi Rgo 61y Zeg one 5 10 fifteen Tht Suz Agd Hei Ahd SHi Tug Tug Azr 51p Tht A1a SHP month Suz Suz twenty 25 thirty Zeg HOUSE Suz Zeg Rgo C1u SHP NHC A1a Bz Wah Pb Suz Tht HOUSE Tht 35 40 45 Zeg Azr Tht Wah! Suz Azr Zeg Suz (Pi Azr Zeg Tht Tug YOU C1p Hei fifty 55 60 Tgr Azp thr V "! Rgo C1 and Suz Hei Zeg Suz C1u Zeg Agd Suz Zeg Zeg 65 70 75 80 Azr 01p Wah C1 and Tht C1p A1a Suz Tht Agd SHi C1p Azp Agd Not Suz 85 90 95 Tht Suz Ahd Rgo C1u Tgr Tug Suz A1a Hei Zeg Bz <31p C1 and C1u Suz one hundred 105 110 Ahd Hei Suz Aha Rgo Hei Agd HOUSE Suz Agd Rgo SHU Pb C1u Wah Aha 115 120 125 Agd Rgo SHU Tht C1 and Tht Zeg Azr Ya1 Wah! Suz Bz Rgo Suz A1a Rgo 130 135 140 C1u Tht Pb Zeh Azp Tht Tht Zeg Zeg Tht Azr Not Suz Agd Rgo Nhz 145 X50 155 160 SHP Not Suz Azp Wah Ya1 A1a Not Rgo <31u AZP A1a Zeg Agd Azr A1a 165 170 175 Ya1 Suz Tht Zeg Tht Zeg Rgo Tht Agd Zeg May Aha Rgo C1u Aha Ya1 130 185 190 NHC Hei Rgo C1p Rgo Ya1 Zeg Tht Agd Zeh C1p M1z Tht C1p Rgo Tht 195 200 205 Rgo 01and Rho Zeg Tht A1a Rgo Zeg Tht Zeg Pb Hei Hei Rgo Me C1u 210 215 220 Rgo Zeg Rgo Rho Aha 61and <31u Zeg Tht C1u Azr 225 230 235

(2) ΧΝΓ0ΡΜΑΤΙ0Ν ROCK ZEO TO N0: 36:

(ί) δΕΟϋΕΝΟΕ СНААСТЕК13Т1С5:

(A) EYASTN 4 athpo ACX ^ h (B) ΤΥΡΕ: aa1po askhy (C) 5ΤΒΑΝ0Ε0ΝΕ35: ipkpoyp (O) ΤΟΓΟΧΟΟΥ: ipkpoyp (11) MSKHESPHE ΤΥΓΕ: rgosehp (X1) ZEOTSEKSE 0E5SK1RT1SI: ZEO TO N0: 36

A1a 31l Mek Suz (2) ΙΝΓΟΡΜΑΤΙΟΝ GOK 5E <2 TO N0: 37:

(x) ZEOOEISSE SNAVASTEV15T1SZ:

(A) HEMSTN: 5 APPO ASHCAS (B) ΤΥΡΕ: ATHPO ACCHO (C) 5ΤΚΑΝΟΕΟΝΕ25: IKPOPYP (O) ΤΟΡΟΕΟΟΥ: IKPOPYP (11) MOGIETS р: RGOSEKPP (Ha) ZEOZEISE OEZSKHRYUTY: 37EO N0

THX AXA C1P Meo Souz

5 (2) ΙΝΓΟΗΜΑΤΙΟΝ GOA 5E0 10 N0: 39:

(ί) 3ΕΟϋΕΝΟΕ SNACASTEA15T1S5:

(A) LIST: b а п ас х (((((B) ΤΥΡΕ: х х о ас х <<<1 (C) 3ΤΚΑΝΟΕΟΝΕ53: кккпып (()) ΤΟΡΟΧΟΟΥ: ккк МО (() (11) MOSCOW ΤΥΡΕ ': гососехпп ((!!) 3ΕαϋΕΝ0Ε 0Ε30ΚΙΡΤΙ0Ν: 5Е0 THAT: 5Е0

SHHP THX AXA SHHP Mek Suz

5 (2) ΙΝΓΟΡΜΑΤΙΟΝ GOA 5E0 TO N0: 39:

(1) δΕΟϋΕΝΟΕ SNACASTEC15T1SZ: (Α) ΧΕΝΟΤΗ: 7 at1po ac! (3c (B) ΤΥΡΕ: at1po ac! <1 (C) 5ΤΚΑΝΟΕΟΝΕ55: ipkpoip (O) SPEED: ipkpoip

109

110 (ϋ) MILLION ΤΥΡΕ: rgosekhp (xx) ZEOCEISE 0Ε3ΟΗΙΡΤΙΟΝ: WB ΙΟ N0: 39

Azr 61η ΤΪ1Γ A1a 61o Mae Suz

5 (2) ΙΝΓΟΒΜΑΤΙΟΝ GOK ZEO 10 N0: 43:

(1) ZEOEMSE SYANASTEYAHZT1SZ:

(A) GEM: 11 Ash Chlo AskhDz (B) ΤΥΡΕ: Ahhh Askhs!

(C) 3ΤΗΑΝ0Ε0ΝΕ33: ipkpoip (O) Τ0Ρ0106Υ: ipkpoip (11) MESSECO ΤΥΡΕ: rgosekhp (2) ΙΝΪΌΗΜΑΤΣΟΝ GON ZBO Yu N0: 40:

(1) ZEOEISSE SNACASTEA15T1SZ: (Α) 1ΕΝ6ΤΗ: 8 ххп ас асх (B B 8 8ппп (((((((((пп (п (пп ((п (пп (п (((ппп ((( 40

Tug Azr 61p Tg A1a C1p Mee Suz

5 (xx) 5Е0ОЕИСИС 0Ε3ΟΗΙΡΤΙΟΝ: ЗЕО 10 N0: 43:

Oh? <31 and Tue Tug Azr <31n Tg A1a S1n Mes Suz

10 (2) ΙΝΓΟΡΜΑΤΖΟΝ GOK ZEO Σ0 N0: 44:

(1) ZEOCEXB SNNLSTEIA15TKhSZ: (Α) 1ΕΝ0ΤΗ: 12 ASHPO ASHYZ (B) ΤΥΡΕ: AAHPO AS14 (C) 3ΤΗΑΝΟΕΟΝΕ53: IKPOIP (O) ΤΟΡΟίΟΟΥ: IKKPOIP (ϋ) MOSIBE ΤΥΡΕ: ROGOEK (2) ЗОГЕХ (0) :

X OEZSNHRTYU: ZBO 10 N0: 41

Tue Tue Azr 6Hp Tg A1a S1p Mes Suz

5 (X1) ЗЕОиЕИСЕ 0Ε30ΗΙΡΤΙΟΝ: ЗЕО 10 N0: 44:

Bey Ag? 01i Tug Tug Azr S1p TNg A1a 01p Mes Suz $ 10 (2) ΙΝΕ0ΚΜΑΤΙ0Ν GON ZBO 10 N0: 45:

(ί) ZEOEXE SNAYAASTEVHZT1C5:

(A) EMZTYA 13 ashhpo APA of (B) ΤΥΡΕ:! Aahpo ac d (C) 3ΤΗΑΝΟΕΟΝΕ33:! Ipkpoip (ϋ) Τ0Ρ0ΙΛ0Υ: _h ipkpoip (11) MOESiE ΤΥΡΕ: rgosehp (2) ΙΝΓΟΗΜΑΤΙΟΝ ΓΟΗ FRL GO N0: 42:

(ί) ZEOCEISE SNANASTEN13T1C8: (Α) & ΕΝ6ΤΗ: 10 at1po ac1 <1z (B) ΤΥΡΕ: aa! ZESIEKSE 0EZSN1RT10Y: ZEO 10 N0: 42:

61 and Tue Tue Azr 61p Tg A1a 61n Mes Suz 15 10 (x!) 8Ε0υΕΝΟΕ 0Ε30ΗΣΡΤΙ0Ν: ЗЕО 10 N0: 45:

Huh? Oh oh 01 and Tug Tug Azr 01p Thx A1a 61p Mes Suz 15 10 (2) ΙΝΕ0ΒΜΑΤΙ0Ν GON ZBO 10 N0: 46:

(1) ZEOOMSE SNAYASTAYA13T1SZ:

(A) LITTLE: 14 ashhpo as1c1z (B) ΤΥΡΕ: ashhpo as14 (C) 3ΤΗΑΝ0Ε0ΝΕ33: ipkpoip (O) ΤΟΡΟΙΌΟΥ: ipkpoip (11) MOSESSES ΤΥΡΕ; rgosekhp -.

(x1) ZEOTEX 0Ε3ΟΗΖΡΤΧΟΝ: ZEO GO N0: 46:

Suz Ag? Sai Ag? C1 and Tug Tug Azr <31p tye A1a 31p Mae Suz 15 10 (2) ΙΝΤΟΚΜΑΤΣΟΝ ROCK ZEO GO N0: 47:

(1) ZEOSEIS SYAJAASTEKHZTHHSZ:

(A) SEISTN: 15 ashkhpo askhskhz (B) ΤΥΡΕ: ashkhpo askhO (C) 3ΤΚΑΝΟΕΒΝΕ83: ipkpoip (O) ΤΟΡΟΕΟΟΥ: ipkpoip (XX) MOSCESSE ΤΥΡΕ: rgosekhp (xx) ZEOKHMSE ΟΕδΟΒΙΡΤΧΟΝ: ZEO 10 N0: 47:

Thz Suz Le? Sai Ae? C1 and Tug Tug Azr <31p TNg A1a 01p Mes Suz 15 10 15 (2) ΙΝΕΟΚΜΑΤΧΟΝ ΕΟΚ ZEO GO N0: 48:

(1) 3 £ <3ϋΕΝΟΕ SNANASGEKKHZTKhSZ: (Α) ΙΧΝΟΤΗ: 16 ASHPO ASKHOZ (B) ΤΥΡΕ: ASKHPO ASKHY (C) 3ΤΗΑΝΟΕΟΝΕ33: IKKPOIP (s) ΤΟΡΟΟΟΟΥ: IKKPOIP (ϋ) MYESISE τυρέ: РГЕЗЕЗЕ ( GO N0: 48:

Zeg Tje Suz Ae? Bey Ag? <E1i Tug Tug Azr C1p TLg A1a 01p Mes Suz 1 5. 10 15 (2) ΙΝΕΟΗΜΑΤΙΟΝ GOK GO 3 10 N0: 49:

(x) ZEOTSEISSE SNANASTBAKHZTHHSZ:

(A) SEXT: 17 ash1po askhoz (B) ΤΥΡΕ: ashkhpo askh (C) 8ΤΚΑΝΟΕΟΝΕ33: ipkpoip (O) ΤΟΡΟΧΟΟΥ: ipkpoip (ϋ) MOSSESS ΤΥΡΕ: rgosekhp

111

112 (2) (2) (2) (χχ) ZETSECYSE 0 EZSKHRT10Y: WEIGHT GO N0: 49:

С1у Вех Тхх Суз Агд Лей Ahd С1и Тух Туг 15 10

Suz

ΙΝΓ0ΗΜΑΤΙ0Ν GOK ZEO £ 0 N0: 50:

(ί) (xx)

Rgo

Month

Azr C1p

Tht

A1a

С1п mon (2) (ί) (ϋ) δΕΟϋΕΝΟΕ СЯАЯАСТЕК13ТХСЗ:

(A) (B) (C) (O)

LF: 19 APHPO ASHYZ ΤΥΡΕ: APHPO ASHY δΤΚΑΝϋΕϋΝΕδδ: IPKOPYOP TOROYOSU: IPKPOP

MOGIETS ΤΥΡΕ: rgosexp δΕΟϋΕΝΟΕ 0Ε50ΚΙΡΤΙ0Ν: ZEO GO N0: 50:

C1u Zeg Tg Suz Ahd Ley Ahd 01i Tug

10

Tug Azr

31p

Tht

A1a C1l (2) (xx)

Rgo

Suz

ΙΝΡΟΚΜΑΤΙΟΝ GOA ZEO GO N0: 51:

(ί) (ϋ) (XX)

LITTLE: 19 APHPO ASHYZ ΤΥΡΕ: ATHPO ASKHO δΤΚΑΝϋΕϋΝΕδδ: IPKOPYOP TOROYOSU: IPKPOP

MOGIETS ΤΥΡΕ: ososekhp δΕΟϋΕΝΟΕ OEZESAHRTHHOY: ZEO GO N0: 51:

(2)

C1 and

C1p

Рgo С1у Зег Тг Suz Ahd Ley Agd C1i

5Tug Tug

Azr

C1p

Tx A1a

May suz

ΙΝΡΟΚΜΑΤΙΟΝ GOA ZEO GO N0: 52:

(1) δΕΟϋΕΝΟΕ СНАААСТЕН13Т1СЗ:

(A) (B) (C) (ϋ)

LITTLE: 20 AP ± AS + 4Z ΤΥΡΕ: ASH1 + AS £ 0 3ΤΚΑΝϋΕϋΝΕ33: IKPOIP TOROYOSU: IKKPOIP (xx) (ϋ) MOGIETS ΤΥΡΕ: PROSEKP δΕΟϋΕΝΟΕ ϋΕδΟΚΙΡΤΙΟΝ: ZEO GO N0: 52:

Рgo 01и Рго 01у Зег Тг Suz Ahd Ley 1 5

Ahd C1i Tug Tug Azr C1p Tg

15 (2)

A1a С1п Мес Суз (2) ΙΝΓΟΚΜΑΤΙΟΝ GOA ZEO GO N0: 53:

(ί) δΕΟϋΕΝΟΕ СНААСТЕАХЗТ1С5:

(A) (B) (C) (ϋ)

SHOPPING: 21 APPPO AC! 4Z ΤΥΡΕ: AP ± ACP 3-333: IPKOPYOP TOROYOSU: IPKPOIP (xx)

MOGIETS ΤΥΡΕ: rgosekhp (xx) δΕΟϋΕΝΟΕ 0Ε50ΗΙΡΤΙ0Ν: ZEO GO N0: 53:

A1a

Rgo C1i Rho C1u Zekh Tg Suz Ahd Ley Agd C1i Tug Tug Azr C1n '5 10 15 (2)

Tht

A1a C1p Mes Suz (2) ΙΝΓΟΚΜΑΤΙΟΝ GOA ZEO GO N0: 54:

(x) δΕΟϋΕΝΟΕ СНААСТЕК13Т1СЗ:

(A) (B) (C) (O)

FAMILY: 22 atpo ashyz ΤΥΡΕ: aapo at ashy 3ΤΚΑΝΟΕΟΝΕ33: ipkopyop TOROYOSU: ipkpoip (xx)

MOGIETS ΤΥΡΕ: rgosekhp (x!)

ZEOCEJAS ϋΕδΟΚΙΡΤΙΟΝ: ZEO 10 N0: 54:

(2)

С1п Тьг АХа С1п Мес Суз δΕΟϋΕΝΟΕ СНАЛАСТЕ13Т1СЗ:

(A) (B) (C) (O)

Lagatk: 23 atppo as ± 4z ΤΥΡΕ: at! At as! 4 3ΤΚΛΝ0Ε0 и53: ipkopyp TOROYOSU: ipkpyop

MILLION ΤΥΡΕ: rgosekhp

ZEOOEYAS OEZSAHRTYUA: ZEO GO N0: 55:

Tug A1a Rgo C1 and Rgo C1u Zeg Thx Suz Ahd Ley Agd C1i Tug Tug 5 10 15

Azr

С1п Т1г A1а С1п Мес Суз

ΙΝΕ0ΚΜΑΤ10Ν GOA ZEO 10 N0: 56:

(I) (ϋ) δΕΟϋΕΝΟΕ СНААСТЕЯ13Т1СЗ:

(A) (B) (C)

YU)

LIST: 24 atpo as! <1Z TOUR: atpo ashy δΤΚΑΝϋΕϋΝΕδδ: ipkopyop TOROOSU: ipkpoip

MILLION ΤΥΡΕ: rgosekhp

ZEOCEMSE 0ЕЗСК1РТ10Ы: ЗЕО 10 N0: 56:

Tht

Tug

Azr S1p Thg A1a 01l Mes Suz

ΙΝΡΟΚΜΑΤΙΟΝ GOA'ZEO GO N0: 57:

(x) (ϋ) (xx) δΕΟϋΕΝΟΕ СНАААСТЕЯ 13Т1СЗ:

(A) LEFT: 25 APHPO ACHLZ (B) ΤΥΡΕ: APHPO ACX4 (C) δΤΚΑΝϋΕϋΝΕδδ: IKPOIP (ϋ) TOROYOSU: IKPOIP

MOGIETS ΤΥΡΕ: rgose1p δΕΟϋΕΝΟΕ ϋΕδΟΚΙΡΤΙΟΝ: ZEO GO N0: 57:

Pb

Tug (1) (11) (xx)

A1a

C1 and

Tg Rgo Tug A1a Rgo C1i Rho C1u Zeg Tg Suz Agd 5 10

Tug Azr C1p Thg A1a C1p Mes Suz

25 δΕΟϋΕΝΟΕ SNAYASTEIA13T1SZ:

(A) (B) (C) (O)

GEMSTN: 26 aShpo as! 4z ΤΥΡΕ: aphpo as! Th 3ΤΚΑΝ0Ε0ΝΕ33: ipkpoip TOROOSU: ipkpoip

MOGIETS ΤΥΡΕ: rgosehp

ZEOOHENS ϋΕδΟΚΙΡΤΙΟΝ: ZEO GO N0: 59:

Ley Agd C1i

Rie Thr Rgo Tug A1a Rgo C1i Rho C1u Zeg Tg Suz Ahd Ley Agd

10 15

Tug Tug Azr C1p Thx A1a C1a Mes Suz '20 23

ΙΝΡΟΚΜΑΤΙΟΝ GOA ZEO GO N0: 59:

(ί) (ϋ) (XX) νβΐ δΕΟϋΕΝΟΕ SNAYALSTEIA13T1SZ:

(A) (B) (C) (O)

HISTORY: 27 APHPO ASLZ ΤΥΡΕ: APHPO AS! <1 δΤΚΑΝϋΕϋΝΕδδ: IKKOPYOP TOROYOSU: IKKPOIP

MOGIETS ΤΥΡΕ: rgosekhp δΕΟϋΕΝΟΕ ϋΕδΟΚΙΡΤΙΟΝ: ZEO GO N0: 59:

A1a RYE YOU

Agd

C1 and Tug Tug

Azr C1p Thg A1a C1p Mes Suz

ΙΝΓΟΚΜΑΤΙΟΝ GOK ZEO GO N0: 60:

(X)

ZEOEEMSE SNAYASTEK13T1S5:

(A) (B) (C) (O)

GEMSTN: 2V APHPO ACX4Z ΤΥΡΕ: Aliao ASX <1 δΤΚΑΝϋΕϋΝΕδδ: IPKOPYOP TOROYOSU: IPKPOIP (ϋ)

MOGIETS ΤΥΡΕ: rgosekhp (x!)

ZEO & VEX ϋΕδΟΚΙΡΤΙΟΝ: ZEO GO N0: 60:

С1п Уа1 A1а Рее Тх Рго Туг А1а Рго С1и Рго С1у Зег Тг Suz Agd 15 ju 15

Lye Ahd S1i Tus Tug Azr S1n Tg A1a S1l Mes Suz 20 25

113

114 (2) ΙΝΓΟΗΜΑΤΙΟΝ GOA ZEO 1 ° N0: 61:

(1) 5Ε0ϋΕΝ € Ε SNAYAASTEYAHZTHSZ:

(A) 1ΕΝ6ΤΗ: 29 al £ ao aXX4z (B) ΤΥΡΕ: aaOao aXy (C) WRITING: ккпыпып (О О О Оп: пкко (((О:: пккоыпып (((Ϊ1) MOGIETЕ ΤΥΡΕ: госеХХпп (2) ΣΝΓΟΒΜΑΤΙΟΝ GOK ZE:

i) ZEOEISSE SJAIASTEK13T1C5: (L) EXT5TN: 7 introduced zsayz (B) ΤΥΡΕ: atKhpo ashy (C) 3-33: unkopyp (V) ΤΟΡΟΙΛΟΥ: unkpoyp (ϋ) MOSYOZE OX: OXYZOZEZZYZZZYZZYOZZYPZOZZYZ (10) :

A1a С1п Уа1 АХа Ре ТЬг Рго Туг АХа Рго <Зхи Рго С1у Зег Тгг Суз 15 1015

Agd Ley Agd S1i Tus Τνε Azr 6Hp Thg Aha S1p Mes Suz ’25 (xx) ZEOTSE'SE 0EZSV1RT10Y: ZEO GO N0: 67

AXa Rgo Lei Agd Luz Suz Agd (2) ΙΝΓΟΗΜΑΤΙΟΝ GOA ZEO 10 N0: 62:

(X) ZEOTSEMSE SNAYAASTEYAHZTHSZ:

(A) LITTLE: 30 atxpo asX4z (B) ΤΥΡΕ: aaXpo asXO (C) 5 :53: ipkpoip (O) ΤΟΡΟΙΟΟΥ: ipkpaip (XX) MILLION ΤΥΡΕ: rgohexn (2) ΙΝΓΟΚΜΑΤΙΟΝ GOK ZEO GO N0: 68:

(X) ZEOEMSE SNAKASTEK13T1S8: (Α) LISTN: 31 Base Razhz (B) ΤΥΡΕ: PIS1EXS ASX4 (C) 5ΤΚΑΝΒΕΒΝΕ33: IKPOPYEP (B) HEARNESS: IKPOPYEP (ϋ) MOSTER EXTERNAL 62:

Рgo АХа (ЗХп УАХ АХа РЬе Тгг Рго Туг А1а ₽iseo СХи Ρεο СХу Зег Тгг 15 Ю 15

Suz Agd Hei Agd S1i Tug Tug Azr S1p Tg AHa SHHp Mes Suz

25 30 (2) ΙΝΓΟΗΜΑΤΙΟΝ GOA ZEO 10 N0: 63:

(ί) ZEOTSEISSE SNAYAASTEYA13T1S5:

(A) BYBEST: 31 atHpo asKh03 (B) ΤΥΡΕ: atKhpo askh (C) 3-35: ipkpoyp (O) ΤΟΡΟΧΟΟΥ: ipkpoyp (XX) MOSYOE ΤΥΡΕ: rgosekhp (xX)

Chai rgo AXa S1p Ya1 AXa Rie Thb Rgo Tug AXa Rgo SXi Rgo C1u Zeg 15 10 15

Tg Suz Agd Ley Agd SHi Tug Tug Azr S1n Tg AhA SHhp Mes Suz

25 30 (2) ΙΝΤΟΗΜΑΤΙΟΝ GOA ZEO 10 N0: 64:

(1) ZEOTSEISE SNAYAASTEYAHZTHSZ: (λ) ΧΕΝΟΤΗ: 4 atHpo asHbz (B) ΤΥΡΕ: atHpo asHO (C) 3ΤΗΑΝΟΕΟΝΕ55: ipkpoyp (O) Bull: ipkpoyp (XX) M0HESKH1HE ΤΥΡΕ: rgoseHp (xX) ZEOTSEMSE OEZSYAHRTHOY: ZEO 10 N0: 64

AXa Rgo Hei Agd (2) ΙΝΓΟΗΜΑΤΙΟΝ GOA ZEO 10 N0: 65:

(X) 3Ε0ν £ Ν0Ε СНАААТАЕХЗТХСЗ:

(A) HYSTN: 5 ATHPO ACHDZ (B) ΤΥΡΕ: ATKHPO ASKHO (C) WRONG: IKKOPYOP (□) ΤΟΡΟΧΟΟΥ: IKKOPYP (XX) MOSHEKHE ΤΥΡΕ: PROSEKP (XX) ZEOZEES ΤΙΟΝ 0: 3 Ε 0: 3

SSTTASSSSAT ATSSASASSSS TTTSSSSSSSSA (2) ΣΝΓΟΒΜΑΤΙΟΝ GOK 5E0 10 N0: 69:

(x) ZEOCEMSE SNACASTEA15T1SZ: (Α) LIECOTN: 33 Base Razhz (B) ΤΥΡΕ: Pis1ekhs Askh <1 (C) 3ΤΚΑΝΒΕΒΝΕ33: Ipkpoyp (ϋ) ΤΟΡΟΧΟΟΥ: Ipkpoyp (ίί) MOSCES ΤΥΡΕ: OEZE (0) N0: 69

SSSAASSTTT TASASASASS AATTSAAOS STS (2) ΙΝΓΟΚΜΑΤΙΟΝ GOK ZEO Σ0 N0: 70:

(ί) ZEOCEMS SNAKASTEV15T1S5:

(λ) LEFT: 40 Lase rachs {EH ΤΥΡΕ: pisHeHs ashy (C) 3–35: ipkpoip (O) TOPOOST: ipkpoip

I (ϋ) power: τυρέ: οβνα (хх) ЗЕОиЕМСЕ ВЕСЕСАКРТУЫ: ЗЕО ΙΟ N0: 70;

ASTC5AOSAT SSSSbSATAA ATLASTAASS ATSSSSSTSS (2) ΙΝΓΟΚΜΑΤΙΟΝ GOK ZEO 10 N0: 71:

(ί) ZEOTSEMSE SNAKASTEK13T1SZ: (Α) ΙΕΝΟΤΗ: 41 азе рахгз (B) ΤΥΡΕ: pisKhekhs askhy (C) 3ΤΚΑΝΒΕΒΝΕ33: ipkpoip (O) TOROOSOU: ipkpoiip (xx) MOSCEPT ΤΥΡΕ: OXY OXY 65

Aha rgo hei agd hue

5 (xi)

SASSTSSSSAT STATSSASSA SAASSASSSS AAAASSTTTT S (2) ΙΝΓΟΗΜΑΤΙΟΝ GOYA ZEO CHI N0: 66:

(X) ZEOTSEISSE SNAYAASTEYAHZTHSZ:

(A) ΧΕΝΟΤΗ: 6 atKhpo asKhBz (B) ΤΥΡΕ: atKhpo asKhS (C) STANDARD:: ккпоып ((()) ΤΟΡΟΧΟΟΥ: ккпоыпып (XX (XX) MOKHOSEH ΤΥΡΕ: госееХп ((2) ΙΝΓΟΚΜΑΤΙΟΝ GOK 5E0 10 N0: 72:

X 66

AXa Rgs Hei Agd Huz Suz

5 (xx) ZEOVEISE ΒΕ30ΚΙΡΤΣ0Ν: ZEO 10 N0: 72

STSTAOOSSAT ATSSASASSSS TTTSSSSSSSSA (2) ΙΝΓΙΝ GOK ZEO ΙΒ N0: 73:

(£) ЗЕОЕХСЕ СНААСТЕК 15ТХСЗ: (Α) ГЕЫСТН: 34 азе рахгз (B) ΤΥΡΕ: пис1ехс асх <3 (C) 3ΤΚΛΝΟΕΒΝΕ55: unicode (O) AXLE: unicode (ϋ)

115

116 (χί) ZEYOSHE © Е5С & ХРТ10Н: ЗЕО 10 N0: 73: СССССАТССС ТАТТААТТСА АССАССССАА LAOS (2) ΙΝΓΟΒΜΑΤΙΟΗ GOY ЗЕО X © N0: 74:

(x) ZEOCEXE SNAYAASTEN13T1SZ:

(A) OUESTN: 30 раase rakhgz (B) пис: Х писХХХХс ххх (C (C) 3ΤΗΑΝΟΕΟΝΕ83: кккпош ((()) ΤΟΡΟΟΟΟΥ: ккппош ((ϋ (ϋ) MЬЕСииГиΤΥΡΕ ΤΥΡΕ: οΟΝΑ (xx) 3Ε0ϋΕΝΟΕ ϋΕΝΟΕЗЗЗГРГР::: ZEO I © 0: © N0

SSSSATATOT ATATSSASSSS TSAAAATAAAT (2) ΙΝΓΟΒΜΑΤΙΟ »GOA ZEO GO N0: 75:

(1) ZOOOOTIS SNAASTEN13T1SZ:

(A) EXCHANGE: 33 Lazy rakhgz * (8) ΤΥΡΕ: pisHekhs askh (C) 5ΤΗΑΝ0Ε0ΝΕ33: ipkposhp (O) ΤΟΡΟΙΛΟΥ: ipkposhp (xx) MOSES ΤΥΡΕ: οΟΝΑ (xx) δΕΟϋΕΝΟΕ ΟΕ308Ι? ΤΙΟΝ: 75ЕО

SSSAASSTTT TASASAOOASS AATTOAASSA STS (2) ΙΝΓΟΡΜΑΤΙΟΝ GOYA ZEO 10 N0: 76:

(x) ZEOOEEISE SNAYAASTEYAH5T1SZ:

(A) LENOT: 38 Lazer! Sz (8) ΤΥΡΕ: pis1ekhs askh.

(C) 5Τ8ΑΝ0Ε0ΝΕ35: ipkposhp (O) ΤΟΡΟΟΟΟΥτ- ipkposhp (XX) MOSES ΤΥΡΕ: οΟΝΑ (χΐ) ZEOEMSE 0ЕЗСК1₽ТХ0М: ЗЕО 10 N0: 76:

SSSSATATOT SOATTASSTO TASSAAOTSS SASAAASO (2) ΧΝΓΟΚΜΑΤΙΟΝ GOK ZEO 10 N0: 77:.

(1) VEOOTHS SMLCASTEC13T1C5:

(A) LIST: 33 Lazy rahzz '(B) ΤΥΡΕ: write! With ace! 4 (C) 3ΤΚΑΝΟΕΟΝΕ55: ipkposhp (O) Τ0Ρ01Λ6Υ: ipkposhp <xx) MYÖZSCHE ΤΥΡΕ: SOYA (xx) ZEOZENESE ALL: 5: 0 :

SSSAASSTTT TASASASASS AATTZAASS STO 33 (2) ΙΝΓΟΚΜΑΤΙΟΝ GOK 5E0 GO N0: 78:

(ί) ZEOCEISE SNAYAASTEKHZT1C5:

(A) ΕΕΝΟΤΗ: 30 азase rahhz - (B) пис: пис Х с с с ас х C (C) 3 )33: ipkposhp (ϋ) ΤΟ? ΟΙ.Ο <3Υ: ipkposhp. . (ϋ) MOSCOW ΤΥΡΕ: οΟΝΑ (хх) 3ΕΟΟΕΝΟΕ 0 ЕЗСК1РТ10Ы: ЗЕО ΙΟ N0: 79:

SSSSATATOT STASSAASTS SSASAAASSA 30 (2) ΙΝΓΟΚΜΑΤΙΟΝ GOA ZEO 10 N0: 79:

(x) 3Ε0ϋΕΝΟΕ S. CHAYALSTEK13T1SZ:

(A) ΙΕΝΟΤΗ: 33 азase rahhz (B) 1: pis1ekhs askhb (C) 3ΤΚΑΝΟΕΟΝΕ33: ipkposhp (O) Τ0Ρ0100Υ: ipkposhp (ϋ) MYOEONX ΤΥΡΕ: οΟΝΑ (xX) ZEOEMSE 0Е5СК1РТЫЫ: ЗЕО 10 N0: 0 ЗЕО 10 N0: 0

SSSAASSTTT TASASASASS AATTOAAOS STS 32 (2) ΙΝΓΟΚΜΑΤΙΟΝ GOK ZEO 10 N0: 80:

(x) ZEOSEYAS SNAYAASTEK15T1S5:

(A) Χ.ΕΝ6ΤΗ: 31 азaze rakhgz (B) ΤΥΡΕ: pisHekhs askhb (C) 3ΤΚΑΝΟΕΟΝΕ33: ipkposhp (ϋ) ΤΟΡΟΙΌΟΥ: ipkposhp (xx) MOOESECIE ΤΥΡΕ: ΟΟΝΑ (Χί) SESSENSE ОЕСЯСХАРТХОМ: 5: 0 10

6STTASSSSAT ATSSASASSSS TTTOSSSSSSA A 31 (2) ΧΝΓ0ΗΜΑΤΙ0Ν GOK 8ES GO N0: 81:

(x) ZEOOEMSE SNAJASTEYAH5THSZ:

(A) ΙΧΝ0ΤΗ: 33 Base Razhz (B) ΤΥΡΕ: 11 1 1s ASU (C) 2ΤΚΑΝΟΕΟΝΕ53: кккошош (О (O) SPEED: пккошош ((Х Х (X1) MOSESPХЕ ΤΥΡΕ: οΟΝΑ (xx) ZEOEMEΟΕΕΟΚΙΡΤΙΟΝ ΟΕΕΟΚΙΡΤΙΟΝ: 5Е0 ΙΟ N0: 81:

SSSAASSTTT TASSTSSASA SSSTSTTSTS TTT 33

Claims (47)

1. A truncated tumor necrosis factor (κΤΝΡΈ) receptor having the following formula: K1- [Suk ^19- Suk ¹⁰³ ] -K2, where [Suk ^19- Suk ¹⁰³ ] represents the amino acid residues 19 through 103 of the κΤΝΡΗ-Ι sequence of Betz ΙΌ ЫО : 2 shown in FIG. 1, K denotes a methionylated or non-methionylated amino group of Souk ¹⁹ , or an amino-terminal amino acid residue, or a sequence selected from the group including FROM 1C 81C N810 (ZEO ΙΌ N0: 15) ΝΝ8ΙΟ (8Εζ) Ιϋ N0: 16) 0ΝΝ8ΙΟ (8Εζ) Yu N0: 17) ΡΟΝΝδΙΟ (8Εζ) Ιϋ N0: 18) ΗρςΝΝδίο (Zeo Yu N0: 19) ΙΗΡ0ΝΝ8ΙΟ (8Εζ) Ιϋ N0: 20) ΥΙΗΡζ) ΝΝ8Ι0 (8Εζ) Ιϋ N0: 21) ΚΥΙΗΡςΝΝδΙΟ (8Εζ) Yu N0: 22) ΟΚΥΙΗΡζ) ΝΝ8ΙΟ (8Εζ) ΙΌ N0: 23) ςΟΚΥΙΗΡΟΝΝδΙΟ (8Εζ) Ιϋ N0: 24) ΡςΟΚΥΙΗΡΟΝΝδΙΟ (8Εζ) ΙΌ N0: 25) ΟΡΟΟΚΥΙΗΡΟΝΝδΙΟ (8Εζ) ΙΌ N0: 26) νΟΡςΟΚΥΙΗΡΟΝΝδΙΟ (8Εζ) Ιϋ N0: 27) ενορςοκγΐΗΡΟΝΝδίο (8Εζ) Yu N0: 28) ϋδνΟΡςΟΚΥΙΗΡΟΝΝδΙΟ (8Ε <2 S N0: 29), and K ₂ denotes a carboxy group of Suk ¹⁰³ , or a carboxy-terminal amino acid residue, or a sequence selected from the group G ΡΝ ΡΝΟ YESC5 (8Εζ) Ιϋ N0: 30) ΡΝ08Ι. (8Ε0 Yu N0: 31) TOSZS (8Ε <2 S N0: 32) ΡΝΟδΕΟί (8Εζ) Ιϋ N0: 33), or its variants and derivatives obtained as disclosed in the description, and when K1 denotes a methionylated or non-methionylated amino group of the amino acid sequence USRROKUSHRRMIBGS or its Ν-terminal truncated variant by 1-15 residues, then I1- [Suk ^19- Suk ¹⁰³ ] -I2 is not additional variant with the formula K1- [Suk ¹⁹ Suk ¹⁰³ ] -ΡССС ^ С ^ -Зз, where К3 denotes carboc117 ACP 118 ^{w 161} -Akp strong group of the amino acid sequence shown in FIG. 1, or a truncated carboxy-terminal portion of an embodiment of the sequence Akp ^111- Akp ^{161 of} FIG. one. 2. A truncated tumor necrosis factor receptor according to claim 1, selected from the group including 3ΤΝΓΚ-Ι 2.6О / СУ5 рИ2-MPZUSR <ZAKU1NR0ZH31S- (Su ₃ ¹⁵ -Су51 ° '> - RS-СУН], 3ΤΝΈΒ.-Ί 2.6О / СУ6 [МН2-М03УСРЦСК ¥ 1НРС№С31С- [Суз'Э-Суз ^ З ] EUSZSOOOI], 5ΤΝΡΚ.-Ι2.6Ο / Ν103 [ΝΗ2-Μη5νθΡΟΟΚ ¥ ΙΗΡΰΝΝ5ΙΟ [Suz'Z-Suz'OZ ^ W-COOH], 5ΊΝΕΚ-Ι2.3П / 48 [IN2-MUSHR (2M№1S- [Su519 Suz ¹⁰ 3] - ™ cXB-COOH], zTMKK-12.30 / 415 [MH2-M313- [Suz ¹⁹ -Su ₅ 103]. NGZZZ-UNO] that 5ΤΝΡΚ-12.30 / 018 [ING-MCHSuz ^ -Suz! ⁰³ 1-EN'CZH UNO]. 3. A truncated tumor necrosis factor receptor (kTJAEK) having the following formula: K.4- [Suk ^32- Suk ¹¹⁵ ] -K_5, where [Suk ^32- Suk ¹¹⁵ ] are amino acid residues from Suk ³² to Suk ^{115 of the} sequence 8EC ΙΌ N0: 35 shown in FIG. 8, K ₄ denotes a methionylated and non-methionylated amino group of Suk ³² or an amino-terminal amino acid residue or a sequence selected from the group consisting of ms Compulsory medical insurance A <EMC (8Εζ) GO N0: 36). TAOMS (8E0 GO N0: 37). OTAS) MS / 5E0 GO N0: 38). OOTAOMS (5E <? GO N0: 39). UOOTAOMS (8Εζ> GO N0: 40). ΥΥΌΟΤΛΟΜΟ (5Е0 Ю N0: 41). EUWORTLOMS (8Е <2 GO N0: 42). EGOUUGUTLOMS (8Εζ> GO N0: 43). ΕΚΕΥΥΟΟΤΛΟΜΟ (8Е0 GO N0: 44). KKEUUOOTOAOMS (8Е0 GO N0: 45). SKKEUUOOTOAOMS (8Εζ) GO N0: 46). TSKKEUUOOTAAOMS (8Е0 GO N0: 47). ZTSKKEUUOOTAAOMS <8 EU) GO N0: 48). ΟδΤΟΚΕΚΕΥΥϋΟΤΑΟΜΟ (ZEO GO N0: 49). ΡΟΧΤΟΚΕΚΕΥΥΙΧ, ιΤΛΟΜΟ (8Е <_) GO N0: 50). EROZTSKKEUUOOOOTAOMS (8Εζ> GO N0: 51). RERO8SKKEUUOUTA <2MS (8E <2 GO N0: 52). ARER08TSKEKEUUO <2TA <EMC (8E <E GO N0: 53). ΥΑΡΕΡΟδΤΟΚΕΚΕΥΥϋΟΤΑΟΜΟ (8Εζ> GO N0: 54). RUARERS8TSKKEUUO <2TAOMS (8E <2 GO N0: 55). TRUARERSZTSKKEUUOOTAAOMS (8Εζ) GO N0: 56). ETRUARERO8TSKKEUUO0TA0MS (8E0 GO N0: 57). ΑΕΤΡΥΑΡΕΡΟδΤΟΚΕΚΕΥΥϋΟΤΑΟΜΟ (8Е <2 GO N0: 58). \ / ΑΡΤΡΥΑΡΕΡ08ΤΟΚΕΚΕΥΥΌΟΤΑΟΜΟ (5Е <? ГО N0: 59). OUAETRUAREROZERZUUUOOOOTAOMS (8E0 GO N0: 60). А0УУЕТРУАРЭР <Э8ТСШ.КЕУУООТА <EMC (8Е <2 ГО N0: 61). ΡΑςνΑΕΤΡΥΑΡΕΡΟδΤΟΚΕΚΕΥΥϋΟΤΑΟΜΟ (8Е <2 GO N0: 62). ΕΡΑΟνΑΕΤΡΥΑΡΕΡσδΤΟΚΣΚΕΥΥΟΟΤΑςΜΟ (8Εζ) ГО N0: 63). and K ₅ denotes a carboxy group of Suk ¹¹⁵ , or a carboxy-terminal amino acid residue, or a sequence selected from the group including BUT AR AR ARJE (5Εζ) GO N0: 64) ΑΡ1ΉΚ (5Е <2 GO N0: 65) ARKKS (5E0 GO N0: 66) ARKKSK (8Εζ) GO N0: 67) or its variants and derivatives obtained as disclosed in the description, wherein when K4 is the methionylated or non-methionylated amino group of the amino acid sequence ΤСК ^ КЕΥΥ ^^ ΤΑ ^ ΜС or its ^ terminal truncated by 1-15 residues variant, then K4- [Suk ^32- Suk ¹¹⁵ ] -K5 is not an additional option with the formula K4- [Suk ³² Suk ¹¹⁵ ] -ARKKSK-K6, where K ·, denotes the carboxyl group of the amino acid sequence of Progo ^123- Tyg ¹⁷⁹ shown in FIG. 8, or a truncated carboxy-terminal portion of a variant of the Progo-Tyg sequence shown in FIG. 8. 4. A truncated tumor necrosis factor receptor according to any one of claims 1 to 3, characterized in that it has a non-glycosylated amino acid sequence. 5. A truncated tumor necrosis factor receptor according to any one of claims 1 to 3, characterized in that it has a glycosylated amino acid sequence. 6. A truncated tumor necrosis factor receptor according to any one of claims 1 to 5, characterized in that it is conjugated to a water-soluble polymer. 7. A truncated tumor necrosis factor receptor according to claim 6, characterized in that the water-soluble polymer is polyethylene glycol. 8. A multivalent receptor for tumor necrosis factor containing at least one of the receptors according to any one of claims 1 to 7. 9. The multivalent receptor for tumor necrosis factor according to claim 8, characterized in that it contains a truncated receptor dimer according to claim 3. 10. The multivalent tumor necrosis factor receptor of claim 8, having the formula K.1-XK ₂ , where X is a linker, wherein said linker is a water-soluble polymer, and K and K ₂ are biologically active molecules covalently linked to said water-soluble polymer, despite the fact that at least one of the components K and K ₂ is a truncated tumor necrosis factor receptor according to any one of claims 1 to 5. 11. The multivalent receptor of claim 10, wherein the water-soluble polymer is polyethylene glycol. 12. The polyvalent receptor according to claim 11, characterized in that the truncated receptor is selected from the group consisting of kTKL 2.6 O / C105bb and kTKHEKD 2.6 P / C106bb. 13. Polynucleotide encoding a tumor necrosis factor receptor according to any one of claims 1 to 3, selected from the group including 119 120 polynucleotide with the sequence shown in FIG. 2 (8Е0 ΙΌ НО: 3); polynucleotide with the sequence shown in FIG. 3 (8Е0 ΙΌ НО: 5); polynucleotide with the sequence shown in FIG. 4 (8Е0 ΙΌ НО: 7); polynucleotide with the sequence shown in FIG. 5 (8Е0 ΙΌ НО: 11); polynucleotide with the sequence shown in FIG. 6 (8Е0 ΙΌ НО: 9); polynucleotide with the sequence shown in FIG. 7 (8Е0 ΙΌ НО: 13); polynucleotide with a sequence that is degenerate with respect to the coding regions of these sequences. 14. A vector containing the polynucleotide of claim 13, operably linked to an expression control sequence. 15. The strain of the prokaryotic cell host containing the polynucleotide according to item 13 or the vector according to item 14. 16. The strain of the eukaryotic cell host containing the polynucleotide according to item 13 or the vector according to item 14. 17. A method for producing a truncated tumor necrosis factor receptor, comprising cultivating host cell strains according to claim 15 or 16 under conditions providing for expression of said truncated tumor necrosis factor (δΤΝΡΚ) receptor cell by host cells and isolating it. 18. The method according to 17, characterized in that said host cells are E. soy cells. 19. The method according to 17, characterized in that said host cells are Chinese hamster ovary cells. 20. A truncated tumor necrosis factor receptor according to any one of claims 1 to 7, characterized in that it is obtained by a recombinant method. 21. A method of obtaining a pharmaceutical composition, which consists in the fact that a therapeutically effective amount of a tumor necrosis factor receptor according to any one of claims 1 to 7 or 20, or a polyvalent tumor necrosis factor receptor according to claims 8 to 12, is mixed with one or more pharmaceutically acceptable carriers . 22. A pharmaceutical composition comprising a truncated tumor necrosis factor receptor according to any one of claims 1 to 7 or 20, in combination with a pharmaceutically acceptable carrier. 23. A pharmaceutical composition comprising a multivalent tumor necrosis factor receptor according to any one of claims 8-12, in combination with a pharmaceutically acceptable carrier. 24. The pharmaceutical composition according to p. 22 or 23, characterized in that it is a continuous release composition. 25. The pharmaceutical composition according to any one of paragraphs.22-24, characterized in that it is lyophilized. 26. A method of treating ΤΝΡ-caused diseases, comprising administering to a patient a truncated tumor necrosis factor receptor according to any one of claims 1 to 7 or 20 or a polyvalent receptor according to any one of claims 8- 12. 27. The method according to p, characterized in that the ΤΝΡ-caused disease is diabetes, hyperalgesia, inflammatory bowel disease, ischemic and reperfusion injury or rheumatic diseases. 28. The method according to item 27, wherein the rheumatic disease is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile arthritis, ankylosing spondylitis, dermatomyositis, psoriatic arthritis, scleroderma, Sjogren's syndrome and vasculitis. 29. The method according to any one of paragraphs.26-28, characterized in that the truncated or polyvalent tumor necrosis factor receptor is administered intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly or by infusion. 30. The method according to any one of paragraphs.26-29, characterized in that the truncated or polyvalent tumor necrosis factor receptor is administered before, jointly or after administration of an anti-inflammatory agent. 31. The method according to item 30, wherein the anti-inflammatory agent is selected from the group comprising non-steroidal anti-inflammatory drugs, corticosteroids, slow-acting anti-rheumatic drugs and drugs that modify the disease. 32. The method of claim 30, wherein the anti-inflammatory agent is an interleukin-1 inhibitor (ΙΣ-1) selected from an interleukin-1 receptor antagonist (GB-1ga) or a soluble ΙΠ-1 receptor. 33. The use of a truncated tumor necrosis factor receptor according to claims 1-7 or 20 or a polyvalent receptor according to claims 8-12 for the preparation of a preparation for the treatment of conditioned diseases. 34. The use of a truncated tumor necrosis factor receptor according to claims 1-7 or 20 or a polyvalent receptor according to claims 8-12 for the preparation of a preparation for the treatment of diabetes, hyperalgesia, inflammatory bowel disease, ischemic and reperfusion injuries and rheumatic diseases. 35. The application of clause 34, wherein the rheumatic disease is selected from the group comprising rheumatoid arthritis, osteoarthritis, juvenile arthritis, ankylosing spondylitis, dermatomyositis, psoriatic arthritis, scleroderma, Sjogren's syndrome and vasculitis. 121 122 36. The use according to any one of paragraphs 33-35, wherein said drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intraarticularly or by infusion. 37. The use according to any one of claims 33-36, wherein said drug is administered before, jointly or after administration of an anti-inflammatory agent. 38. The use according to clause 37, wherein the anti-inflammatory agent is selected from the group comprising non-steroidal anti-inflammatory drugs corticosteroids, slow-acting anti-rheumatic drugs and drugs that modify the disease. 39. The use of claim 37, wherein the anti-inflammatory agent is an interleukin-1 inhibitor (GE-1) selected from an interleukin-1 receptor antagonist (ΙΕ-1 ha) or a soluble ΙΕ-1 receptor. 40. A kit for producing aqueous protein compositions comprising a first container with a tumor necrosis factor receptor according to any one of claims 1 to 7 and a second container with a physiologically acceptable solvent. 41. The method according to p, characterized in that the anti-inflammatory agent is methotrexate. 42. The use of claim 38, wherein the anti-inflammatory agent is methotrexate. 43. The pharmaceutical composition according to any one of paragraphs.22-25, characterized in that it further includes an anti-inflammatory agent. 44. The pharmaceutical composition according to item 43, wherein the anti-inflammatory agent is selected from non-steroidal anti-inflammatory drugs, corticosteroids, slowly acting anti-rheumatic drugs and drugs that modify the disease. 45. The pharmaceutical composition according to item 44, wherein the anti-inflammatory agent is methotrexate. 46. The pharmaceutical composition according to item 43, wherein the anti-inflammatory agent is an interleukin-1 (ΙΕ-1) inhibitor selected from an interleukin-1 receptor antagonist D-1ga) or a soluble ΙΕ-1 receptor. 47. The pharmaceutical composition according to item 46, wherein the interleukin-1 receptor antagonist D-1 ha) contains the sequence of the human protein ITA.